Multiplex genome editing of immune cells to enhance functionality and resistance to suppressive environment

ABSTRACT

Provided herein are methods for producing immune cells with disruption of multiple genes. Further provided are methods for inserting a chimeric antigen receptor at a gene locus of an immune cell.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/772,406, filed Nov. 28, 2018, which is incorporated byreference herein in its entirety.

BACKGROUND 1. Technical Field

The present invention relates generally to the fields of immunology,cell biology, molecular biology, and medicine. More particularly, itconcerns multiplex editing of immune cells and methods of use thereof.

2. Description of Related Art

Cellular immunotherapy holds much promise for the treatment of cancer.However, most immunotherapeutic approaches when applied alone are oflimited value against the majority of malignancies, especially solidtumors. Reasons for this limited success include reduced expression oftumor antigens on the surface of tumor cells, which reduces theirdetection by the immune system, the expression of ligands for inhibitoryreceptors such as PD1, NKG2A, TIGIT or CISH that induce immune cellinactivation; and the induction of cells (e.g., regulatory T cells ormyeloid-derived suppressor cells) in the microenvironment that releasesubstances such as transforming growth factor-β (TGFβ) and adenosinethat suppress the immune response and promote tumor cell proliferationand survival. Thus, there is an unmet need for improved methods ofcellular immunotherapy.

SUMMARY

The disclosure provides compositions and methods related to cancerimmunotherapy particularly including engineered immune cells. Specificembodiments concern certain immune cells that have been modified by thehand of man to lack expression of or have reduced expression of one,two, or more genes, and in specific cases the cells with suchmodification(s) also express one or more heterologous proteins,including non-natural proteins such antigen receptors. Also included aremethods of producing the non-natural immune cells. In certain cases, theintroduction of the heterologous antigen receptor is at the genomiclocus of a gene being reduced or eliminated in expression.

In one embodiment, the present disclosure provides an in vitro methodfor the disruption of at least two genes in an immune cell, wherein theat least two genes are selected from the group consisting of NKG2A,SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1,PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40,IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and a combinationthereof. In particular aspects, three, four, five, or six or more genesare disrupted. In specific aspects, the disruption of two or more genesis simultaneous, such as in the same method step. The method maycomprise introducing a guide RNA (gRNA) for each gene to the immunecell.

The method can comprise the knockdown of particular combinations ofgenes, such as the following, for example: (a) NKG2A and CISH, (b) NKG2Aand TGFBRII, (c) CISH and TGFBRII, (d) TIGIT and FOXO1, (e) TIGIT andTGFBRII, (f) CD96 and FOXO1, (g) CD96 and TGFBRII, (h) FOXO1 andTGFBRII, (i) CD96 and TIGIT, (j) CISH and TIGIT, (k) TIM3 and CISH, (l)TIM3 and TGFBRII, (m) FOXO1 and TGFBRII, (n) TIM3 and TIGIT, (o) SIGLEC7and CISH, (p) SIGLEC7 and TGFBRII, (q) CD47 and CISH, (r) CD47 andTGFBRII, (s) SIRPA and CISH, (t) SIRPA and TGFBRII, (u) CD47 and TIGIT,(v) CD47 and SIRPA, (w) A2AR and CISH, (x) A2AR and TGFBRII, (y) ADAM17and CISH, (z) TGFBRII and ADAM17, (a) A2AR and TIGIT, (b) SHP1 and CISH,(c) CISH and TGFBRII, (d) SHP1 and TGFBRII, (e) SHP1 and TIGIT, or (f)SHP1 and TIM3. The method can comprise the knockdown of (1) NKG2A, CISH,and TGFBRII, (2) TIGIT, FOXO1, and TGFBRII, (3) TGFBRII, CD96, andTIGIT, (4) TGFBR2, CISH, and TIGIT, (5) TIM3, CISH, and TGFBRII, (6)CD96, FOXO1, and TGFBRII, (7) TGFBRII, TIM3, and TIGIT, (8) SIGLEC7,CISH, and TGFBRII, (9) CD47, CISH, and TGFBRII, (10) SIRPA, CISH, andTGFBRII, (11) TGFBRII, CD47, and TIGIT, (12) TGFBRII, CD47, and SIRPA,(13) A2AR, CISH, and TGFBRII, (14) TGFBRII, CISH, and ADAM17, (15)TGFBRII, TIM3, and TIGIT, (16) TGFBRII, A2AR, and TIGIT, (17) SHP1,CISH, and TGFBRII, (18) TGFBRII, CISH, and SHP1, (19) TGFBRII, SHP1, andTIGIT, or (20) TGFBRII, SHP1, and TIM3. Any of the above subgroups maybe combined with a second subgroup as disclosed above. For example, anyone of subgroups a-j 1 may be combined with any one or more of the othersubgroups a-j 1, any one or more of subgroups a-j 1 may be combined withany one or more of the other subgroups 1-23, or any one or more ofsubgroups 1-23 may be combined with any one or more of the othersubgroups 1-23.

In some aspects, the method further comprises introducing to the cell anRNA-guide endonuclease, such as Cas9. Introducing the RNA-guidedendonuclease may comprise introducing a nucleic acid, such as mRNA,encoding the RNA-guided endonuclease into the immune cell.

In certain aspects, the immune cell is a T cell, NK cell, B cell,macrophage, NK T cell, or stem cell. In alternative cases, the immunecell is not a T cell, including not a CAR T cell. In some aspects, theimmune cell is engineered to express one or more chimeric antigenreceptors (CAR) and/or one or more T cell receptors (TCR). The immunecell may be virus-specific, such as a virus-specific T cell. The T cellmay be a regulatory T cell. The B cell may be a regulatory B cell. Insome aspects, the stem cell is a mesenchymal stem cell (MSC) or aninduced pluripotent stem (iPS) cell. In particular aspects, the T cellis a CD8⁺ T cell, CD4⁺ T cell, or gamma-delta T cell. The immune cellmay be isolated from peripheral blood, cord blood, bone marrow, or amixture thereof. In some aspects, the cord blood is pooled from 2 ormore individual cord blood units.

In some aspects, an introducing step comprises transfecting ortransducing. For example, introducing comprises electroporation that mayperformed more than once, such as two or three rounds ofelectroporation. In some aspects, a first group of CRISPR gRNAs areintroduced in a first electroporation and a second group of CRISPR gRNAsare introduced in a second round of electroporation. In specific cases,the first group of CRISPR gRNAs are different than the second group ofCRISPR gRNAs. In particular aspects, the first group and/or second groupof CRISPR gRNAs comprise 1, 2, 3, or 4 or more CRISPR gRNAs. In someaspects, two CRISPR gRNAs are introduced in a first electroporation andtwo different CRISPR gRNAs are introduced in a second round ofelectroporation. In specific embodiments, a group of CRISPR gRNAscomprises a group of gRNAs at least two of which target different genes;in particular embodiments, the group of gRNAs each target differentgenes.

In particular aspects, the method comprises disrupting NKG2A, CD47,TGFβR2, and CISH; NKG2A, CISH, TGFβR2 and ADORA2; NKG2A, TGFβR2 andCISH; TIGIT, CD96, CISH, and ADORA2; or ADAM17, TGFβR2, NKG2A, and SHP1.

In some aspects, the disruption results in enhanced antitumorcytotoxicity, in vivo proliferation, in vivo persistence, and/orimproved function of the immune cell. In particular aspects, the immunecell has increased secretion of IFN-γ, CD107, and/or TNFα, compared toin the absence of the modification(s). In some aspects, the immune cellhas increased production of perforin and/or granzyme B, compared to inthe absence of the modification(s).

In additional aspects, the method further comprises introducing a CARand/or TCR to an immune cell, such as introducing a nucleic acidencoding the CAR and/or TCR into the immune cell. In some aspects, thenucleic acid is in an expression vector, such as a retroviral vector. Incertain aspects, the vector is an adenovirus-associated vector, such asAAV6. In some aspects, the vector further comprises an inhibitory genesequence, such as an inhibitory gene sequence selected from the groupconsisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT,CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17,RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5,CD7, and a combination thereof. In particular aspects, the vectorfurther comprises a guide RNA for the inhibitory gene. The CAR may beflanked by homology arms for the inhibitory gene. In some aspects,introducing the vector comprising the CAR sequence results in insertionof the CAR at an inhibitory gene locus, such as an exon of an inhibitorygene, in the immune cell, such that the CAR is under the control of theendogenous promoter of the inhibitory gene. In particular aspects,introducing the vector further disrupts expression of the inhibitorygene.

In another embodiment, there is provided an immune cell, such as animmune cell of the disclosed embodiments, with disrupted expression ofat least two genes in the immune cell, produced at least by the stepcomprising introducing a CRISPR guide RNA (gRNA) for each gene to saidimmune cell, wherein at least two genes are selected from the groupconsisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT,CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17,RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5,CD7, and a combination thereof. In some aspects, three, four, five, orsix or more genes are disrupted.

In certain aspects, the immune cell is a T cell, NK cell, B cell, orstem cell. In some aspects, the immune cell is engineered to express achimeric antigen receptor (CAR) and/or T cell receptor (TCR). The immunecell may be virus-specific, such as a virus-specific T cell. The T cellmay be a regulatory T cell. The B cell may be a regulatory B cell. Insome aspects, the stem cell is a mesenchymal stem cell (MSC) or aninduced pluripotent stem (iPS) cell. In particular aspects, the T cellis a CD8⁺ T cell. CD4⁺ T cell, or gamma-delta T cell. The immune cellmay be isolated from peripheral blood, cord blood, or bone marrow. Insome aspects, the cord blood is pooled from 2 or more individual cordblood units.

In particular aspects, the method comprises disrupting particular groupsof genes, such as NKG2A, CD47, TGFβR2, and CISH; NKG2A, CISH, TGFβR2 andADORA2; NKG2A, TGFβR2 and CISH; TIGIT, CD96, CISH, and ADORA2; orADAM17, TGFβR2 NKG2A and SHP1.

In some aspects, the disruption results in enhanced antitumorcytotoxicity, in vivo proliferation, in vivo persistence, and/orimproved function of the immune cell. In particular aspects, the immunecell has increased secretion of IFN-γ, CD107, and/or TNFα. In someaspects, the immune cell has increased production of perforin and/orgranzyme B.

In some aspects, the cell is engineered to express a CAR and/or TCR. TheCAR may be inserted at an endogenous inhibitory gene locus of the cell,such as and inhibitory gene locus is selected from the group consistingof NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96,ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6,4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, anda combination thereof. In some aspects, the CAR is under the control ofthe endogenous promoter of the inhibitory gene. In certain aspects, theCAR is inserted at the inhibitory gene locus by CRISPR-mediated geneediting.

In some aspects, the CAR comprises an antigen-binding domain selectedfrom the group consisting of F(ab′)2, Fab′, Fab, Fv, and scFv. Incertain aspects, the CAR targets one or more tumor associated antigensselected from the group consisting of CD19, CD319 (CS1), ROR1, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelialtumor antigen, melanoma-associated antigen, mutated p53, mutated ras,HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoproteingp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30,CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambdachain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CD47, CLL-1,U5snRNP200, CD200, BAFF-R, BCMA, CD99, and a combination thereof. Inparticular aspects, the CAR comprises at least one signaling domainselected from the group consisting of CD3, CD28, OX40/CD134,4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70,CD40 and a combination thereof. In some aspects, the immune cellcomprises one or more heterologous cytokines, such as one or more ofIL-7, IL-2, IL-15, IL-12, IL-18, and IL-21. In certain aspects, the CARfurther comprises a suicide gene, such as a membrane bound nonsecretableTNF-alpha mutant or inducible caspase 9.

Further provided herein is an expression vector encoding at least oneCAR and/or TCR, at least one inhibitory gene sequence, and at least onegRNA. In some aspects, the inhibitory gene sequence is from aninhibitory gene selected from the group consisting of NKG2A, SIGLEC-7,LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1,PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R,ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7. In particular aspects, thegRNA is specific to the inhibitory gene. In some aspects, the vector isa viral vector, such as an AAV vector. The CAR may be flanked byhomology arms for the inhibitory gene. Also provided herein is a hostcell, such as a cell of the embodiments, engineered to express thevector of the embodiments. In aspects, the cell is a T cell, NK cell, Bcell, or stem cell.

Also provided herein is a pharmaceutical composition comprising apopulation of immune cells of the disclosed embodiments. Anotherembodiment provides a composition comprising a population of cells ofthe disclosed embodiments for the treatment of an immune-relateddisorder, infectious disease, and/or cancer.

In a further embodiment, there is provided a method of treating adisease or disorder in a subject comprising administering an effectiveamount of immune cells of the disclosed embodiments to the subject. Insome aspects, the disease or disorder is an infectious disease, cancer,such as a solid cancer or a hematologic malignancy, or an immune-relateddisorder. The immune-related disorder may be an autoimmune disorder,graft versus host disease, allograft rejection, or inflammatorycondition, for example. In some aspects, the immune-related disorder isan inflammatory condition and the immune cells have essentially noexpression of glucocorticoid receptor. In certain aspects, the immunecells are autologous or allogeneic with respect to a recipientindividual.

In additional aspects, the method further comprises administering atleast a second therapeutic agent to the individual receiving the immunecells. In some aspects, the at least a second therapeutic agentcomprises chemotherapy, immunotherapy, surgery, radiotherapy, hormonetherapy, or biotherapy. In certain aspects, the immune cells and/or theat least a second therapeutic agent are administered intravenously,intraperitoneally, intratracheally, intratumorally, intramuscularly,endoscopically, intralesionally, percutaneously, subcutaneously,regionally, or by direct injection or perfusion.

Another embodiment provides a method for engineering an immune cell toexpress a CAR comprising using a CRISPR gRNA to insert the CAR at aninhibitory gene locus of the immune cell. In some aspects, the CAR isencoded by an expression vector, such as a retroviral vector, plasmid,lentiviral vector, adenoviral vector, adenovirus-associated viralvector, and so forth. In certain aspects, the viral vector is anadenovirus-associated vector, such as AAV6.

In some aspects, the vector further comprises an inhibitory genesequence, such as an inhibitory gene sequence is selected from the groupconsisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT,CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17,RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7and a combination thereof. In some aspects, the CRISPR gRNA is to theinhibitory gene. In certain aspects, the CAR is flanked by homology armsfor the inhibitory gene. In particular aspects, the CAR is inserted atan inhibitory gene locus at any part of the gene, such as an exon of theinhibitory gene. The CAR may be under the control of the endogenouspromoter of the inhibitory gene. In specific aspects, the CAR disruptsthe expression of the inhibitory gene.

In some aspects, the CAR targets one or more tumor associated antigensselected from the group consisting of CD19, CD319 (CS1), ROR1, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelialtumor antigen, melanoma-associated antigen, mutated p53, mutated ras,HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoproteingp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30,CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambdachain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CD47, CLL-1,U5snRNP200, CD200, BAFF-R, BCMA, CD99, and a combination thereof. Inparticular aspects, the CAR comprises at least one signaling domainselected from the group consisting of CD3, CD28, OX40/CD134,4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70,and CD40. In some aspects, a vector that encodes the CAR also encodes acytokine, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or acombination thereof. In alternative cases, the cytokine is on a vectorseparate from the vector that encodes the CAR. In certain aspects, anexpression construct that encodes the CAR further comprises a suicidegene, such as inducible caspase 9 or a membrane bound, nonsecretableTNF-alpha mutant.

Further provided herein is an immune cell with at least one CAR insertedat an inhibitory gene of the immune cell, such as an immune cellproduced by the present methods. Also provided herein is a compositioncomprising a population of immune cells of embodiments of thedisclosure, such as a population of T cells, B cells, NK cells, NK Tcells, macrophages, stem cells, mixture thereof, and so forth.

Another embodiment provides a composition comprising a population ofcells of the embodiments, and in certain embodiments the population isutilized for treatment of a medical condition of any kind, including atleast for the treatment of an immune-related disorder, infectiousdisease, and/or cancer.

A further embodiment provides a method of treating a disease or disorderin a subject comprising administering an effective amount of immunecells of the embodiments to the subject. In some aspects, the disease ordisorder is an infectious disease; cancer, such as a solid cancer or ahematologic malignancy; and/or an immune-related disorder. Theimmune-related disorder may be an autoimmune disorder, graft versus hostdisease, allograft rejection, and/or inflammatory condition, in somecases. In some aspects, the immune-related disorder is an inflammatorycondition and the immune cells have essentially no expression ofglucocorticoid receptor. In certain aspects, the immune cells areautologous or allogeneic with respect to a recipient individual.

In additional aspects, the method further comprises administering atleast a second therapeutic agent to an individual. In some aspects, theat least a second therapeutic agent comprises chemotherapy,immunotherapy, surgery, radiotherapy, hormone therapy, or biotherapy. Incertain aspects, the immune cells and/or the at least a secondtherapeutic agent are administered intravenously, intraperitoneally,intratracheally, intratumorally, intramuscularly, endoscopically,intralesionally, percutaneously, subcutaneously, regionally, or bydirect injection or perfusion. The immune cells and the at least asecond therapeutic agent may be administered at the same time or atdifferent times, and when they are administered at different times or atthe same time but not in the same formulation, they may or may not beadministered by the same route.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating particular embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: CRISPR/Cas9 mediates efficient multiple genes (NKG2A, CD47,TGFBR2, and CISH) disruption in NK cells. In this set of genes, NKG2Aand CD47 were knocked out in the first round of electroporation and inthe second round of electroporation CISH and TGFBR2 were targeted.Knockout efficiency was successfully validated using PCR andflow-cytometry for both rounds of electroporation. The red (peaks on theright) and blue (peaks on the left) histograms in the flow panelsrepresent expression of the protein before and after CRISPR KO,respectively.

FIG. 2: Validation of multiplex gene editing in NK cells using anotherset of genes (TIGIT (T), CD96 (C), CISH (CH), Adenosine (A)). In thisset of genes, TIGIT and CD96 were knocked out in the first round ofelectroporation and in the second round of electroporation CISH andAdenosine were targeted. Knockout efficiency was successfully validatedusing PCR and flow-cytometry for both rounds of electroporation. The red(peaks on the right) and blue (peaks on the left) histograms in the flowpanels represent expression of the protein before and after CRISPR KO,respectively.

FIG. 3: Disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH) inNK cells leads to enhanced functionality against target tumor cells.There was enhanced IFN-γ, TNFα and CD107 secretion following stimulationwith target cell lines. Flow cytometric analysis of IFN-γ, TNFα andCD107 production was performed with varying NK cells (Edited vs Cas9alone) co-stimulated with target cell lines for 5 hr in the presence ofBrefeldin A.

FIGS. 4A-4B: Disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH)in NK cells leads to enhanced antitumor cytotoxicity. (FIG. 4A) Thecytotoxic activity of gene edited NK cells vs Cas9 only NK cells wasmeasured by ⁵¹Cr-release assay, against K562 (FIG. 4B) Following 30minutes of recombinant TGF-B treatment (50 ng/ml) pSMAD activity wasmeasured by flow cytometry. The addition of exogenous TGF-β failed toinduce activation of pSMAD in the KO CAR-NK cells.

FIG. 5: NK cells lose CD16 and CD62L expression upon cytokinestimulation or target recognition as depicted by CyTOF analysis.

FIG. 6: Knockout of ADAM17 in NK cells prevent shedding of CD16 andCD62L.

FIG. 7: Knockout ADAM17 in NK cells improves ADCC and cytotoxicityagainst K562 targets.

FIG. 8: FACS-based screening of SHP1 knockout efficiency in NK cells at72h.

FIG. 9: Disruption of SHP1 in NK cells leads to enhanced antitumorefficacy. NK cells were co-cultured with K562 or Raji cells at a 1:1ratio for 4 hours. After the incubation, the cells were stained withannexin V and live and dead cells were analyzed. The K562 cells aresensitive to NK cell killing and the Raji cells are resistant to NK cellkilling.

FIGS. 10A-10B: Disruption of SHP1 in NK cells leads to enhancedantitumor efficacy (FIG. 10A). NK cells were co-cultured with K562 orRaji cells at a 2:1 ratio for 5 hours (FIG. 10B). Percent of lysis atvarious effector:target ratios, percentage of IFNγ, TNFα, and CD107a,and percentage of live or dead cells are shown.

FIG. 11: Disruption of SHP1 in NK-CAR cells leads to enhanced antitumorefficacy as assessed by apoptosis assay.

FIGS. 12A-12C: (FIG. 12A) Day 7 FACS-based NKG2A knockout efficiency.(FIG. 12B) Disruption of NKG2A in expanded NK cells leads to enhanceantitumor efficacy. (FIG. 12C) Disruption of NKG2A in NK-CAR cells leadsto enhanced antitumor efficacy against Raji targets.

FIG. 13: The approach was validated with another set of genes—TIGIT (T),CD96 (C), CISH (CH), and Adenosine (ADORA2A) (A). For this set of genes,TIGIT and CD96 were knocked out in one set of NK cells during the firstround of electroporation. CISH and Adenosine (ADORA2A) were targeted forthe second round of knockout in the TIGIT and CD96 KO cells. Knockoutefficiency was successfully validated using PCR and flow-cytometry forboth rounds of electroporation. The red (peaks on the right) and blue(peaks on the left) histograms in the flow panels represent expressionof the protein before and after CRISPR KO, respectively.

FIG. 14: Disruption of multiple genes in NK cells leads to enhancedantitumor efficacy. To evaluate this, multiple gene (NKG2A, CISH,TGFBRII and Adenosine (ADORA2A)) knockout cells and cells electroporatedwith cas9 only were used as the control with K562 (NK sensitive) andRaji (NK resistant) cells for 5 hours. NK cell function was evaluated byflow cytometric measurement and observed increases in TNFα, IFNγ, andCD107a in KO cells upon target cell line stimulation.

FIG. 15: Disruption of multiple genes in NK cells leads to enhancedantitumor efficacy. To evaluate this, multiple gene (NKG2A, CISH,TGFBRII) knockout cells and cells electroporated with cas9 only wereused as the control. NKG2A expression was confirmed by flow cytometry.The addition of exogenous TGF-β failed to induce activation of pSMAD inthe KO CAR-NK cells

FIG. 16: Disruption of multiple genes (NKG2A, TGFβR2 and CISH) in NK-CARcells leads to enhanced antitumor efficacy.

FIG. 17: TGFβR2 KO protects NK-CAR cells from the suppressive effect ofTGFβ.

FIG. 18: Multiplex gene editing is reproducible with different NK-CARconstructs and against different targets.

FIG. 19: Multiplex gene editing of multiple inhibitory genes maintainsNK architecture and protects NK cells from exhaustion induced by TFGβ.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In certain embodiments, the present disclosure provides a novel approachusing CRISPR-Cas9 technology to simultaneously knockdown (or knockout)two or more genes (e.g., genes (such as those listed in Table 1,including adenosine 2a receptor, TGFβR2, NKG2A, TIGIT and/or CISH) inhuman immune cells (e.g., T cells, NK cells, CAR-transduced T cells, orCAR-transduced NK cells). The immune cells may be derived fromperipheral blood or cord blood or a combination thereof.

The present studies demonstrated that decreased expression of theseproteins correlates with improved function, in vivo proliferation andpersistence, and cytotoxicity of T cells and NK cells. This strategyalso protects T cells, NK cells, NK T cells, and iNKT cells from theimmunosuppressive tumor microenvironment, which is mostly driven by TGFβand adenosine. Thus, the present methods can be used to improve theefficacy of various adoptive cellular therapy products (e.g., NK cells,T cells, such as virus specific T cells and regulatory T cells, B cells,such as regulatory B cells, CAR-transduced NK cells, CAR-T cells and TCRengineered T and NK cells, iNKT cells, NKT cells). The adoptive cellulartherapy products may be used to treat various diseases spanning fromcancer (e.g., hematologic or solid malignancies), to infectious diseasesand immune disorders, for example.

In particular embodiments, the immune cells express at least one CAR,and CAR engineering has seen multiple advances in the past few years. Infact, CAR-CD19 has shown impressive clinical results in patients with Bcell leukemia and lymphoma, leading to the FDA approval in two CAR Tproducts in the last year. While CAR-transduced T cells have beenleading the way in the past few years, a panoply of pre-clinical studiesas well as Phase I/II CAR NK trial led by the Applicants have also showneffectiveness of CAR-NK cells against cancer. Despite the advances inCAR engineering, CARs are still mostly transduced into T cells or NKcells using viral vectors, which randomly integrate in the cell's DNAand may result in clonal expansion, oncogenic transformation, alteredtransgene expression, or transcriptional silencing. Thus, finding a wayto target the insertion of the CAR into a specific DNA locus would bevaluable.

Accordingly, in one embodiment, the present disclosure provides methodsfor the insertion of a CAR at a specific gene locus, such as at thelocus of an inhibitory gene or checkpoint protein, using CRISPR/Cas9.The insertion of the CAR at the gene locus can also be used tosimultaneously disrupt expression of the gene, while optionally alsobringing it under the control of the promoter of that gene, whendesired. Specifically, the present methods may direct insertion of a CARat the locus of an inhibitory gene, such as those listed in Table 1,including but not limited to NKG2A, CISH, PD-1, TIGIT, TIM3, SHP1, orTGFβR2, for example using an AAV6 vector and CRISPR/Cas9 technology. Theinsertion of the CAR at the inhibitory gene locus can disrupt theinhibitory effect of a checkpoint molecule (for example), while alsoallowing CAR expression to become under the regulation of the checkpointpromoter and upregulated in the tumor microenvironment. This is usefulfor the applications of CAR therapy in solid tumors, where upregulationof checkpoint molecules can negatively impact the success of CARtherapy. Thus, further methods are provided for producing adoptivecellular therapies, such as T cells, B cells, NK, NKT or iNKT cells,using the CAR insertion method that can have an increased safetyprofile.

I. DEFINITIONS

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. Still further,the terms “having”, “including”, “containing” and “comprising” areinterchangeable and one of skill in the art is cognizant that theseterms are open ended terms. In specific embodiments, aspects of thedisclosure may “consist essentially of” or “consist of” one or moresequences of the disclosure, for example. Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the disclosure. It is contemplated thatany method or composition described herein can be implemented withrespect to any other method or composition described herein. The scopeof the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asused herein, the terms “or” and “and/or” are utilized to describemultiple components in combination or exclusive of one another. Forexample, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone,“x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” Itis specifically contemplated that x, y, or z may be specificallyexcluded from an embodiment.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more. The terms “about”, “substantially”and “approximately” mean, in general, the stated value plus or minus 5%.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present disclosure. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

An “immune disorder,” “immune-related disorder,” or “immune-mediateddisorder” refers to a disorder in which the immune response plays a keyrole in the development or progression of the disease. Immune-mediateddisorders include autoimmune disorders, allograft rejection, graftversus host disease and inflammatory and allergic conditions.

An “immune response” is a response of a cell of the immune system, suchas a B cell, or a T cell, or innate immune cell to a stimulus. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”).

The term “inhibitory gene” as used herein refers to a gene whose geneproduct is directly or indirectly deleterious to the activity,proliferation, and/or persistence of one or more types of immune cells.

An “autoimmune disease” refers to a disease in which the immune systemproduces an immune response (for example, a B cell or a T cell response)against an antigen that is part of the normal host (that is, anautoantigen), with consequent injury to tissues. An autoantigen may bederived from a host cell, or may be derived from a commensal organismsuch as the micro-organisms (known as commensal organisms) that normallycolonize mucosal surfaces.

The term “engineered” as used herein refers to an entity that isgenerated by the hand of man, including a cell, nucleic acid,polypeptide, vector, and so forth. In at least some cases, an engineeredentity is synthetic and comprises elements that are not naturallypresent or configured in the manner in which it is utilized in thedisclosure.

“Treating” or treatment of a disease or condition refers to executing aprotocol, which may include administering one or more drugs to apatient, in an effort to alleviate signs or symptoms of the disease.Desirable effects of treatment include decreasing the rate of diseaseprogression, ameliorating or palliating the disease state, and remissionor improved prognosis. Alleviation can occur prior to signs or symptomsof the disease or condition appearing, as well as after theirappearance. Thus, “treating” or “treatment” may include “preventing” or“prevention” of disease or undesirable condition. In addition,“treating” or “treatment” does not require complete alleviation of signsor symptoms, does not require a cure, and specifically includesprotocols that have only a marginal effect on the patient.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease. Forexample, treatment of cancer may involve, for example, a reduction inthe size of a tumor, a reduction in the invasiveness of a tumor,reduction in the growth rate of the cancer, or prevention of metastasis.Treatment of cancer may also refer to prolonging survival of a subjectwith cancer.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

As used herein, a “mammal” is an appropriate subject for the method ofthe present invention. A mammal may be any member of the highervertebrate class Mammalia, including humans; characterized by livebirth, body hair, and mammary glands in the female that secrete milk forfeeding the young. Additionally, mammals are characterized by theirability to maintain a constant body temperature despite changingclimatic conditions. Examples of mammals are humans, cats, dogs, cows,mice, rats, horses, goats, sheep, and chimpanzees. Mammals may bereferred to as “patients” or “subjects” or “individuals”.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as a human, as appropriate. The preparation of a pharmaceuticalcomposition comprising an antibody or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall aqueous solvents (e.g., water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles, such as sodium chloride, Ringer'sdextrose, etc.), non-aqueous solvents (e.g., propylene glycol,polyethylene glycol, vegetable oil, and injectable organic esters, suchas ethyloleate), dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial or antifungal agents, anti-oxidants,chelating agents, and inert gases), isotonic agents, absorption delayingagents, salts, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, fluid and nutrient replenishers, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart. The pH and exact concentration of the various components in apharmaceutical composition are adjusted according to well-knownparameters.

As used herein, a “disruption” of a gene refers to the elimination orreduction of expression of one or more gene products encoded by thesubject gene in a cell, compared to the level of expression of the geneproduct in the absence of the disruption. Exemplary gene productsinclude mRNA and protein products encoded by the gene. Disruption insome cases is transient or reversible and in other cases is permanent.Disruption in some cases is of a functional or full length protein ormRNA, despite the fact that a truncated or non-functional product may beproduced. In some embodiments herein, gene activity or function, asopposed to expression, is disrupted. Gene disruption is generallyinduced by artificial methods, i.e., by addition or introduction of acompound, molecule, complex, or composition, and/or by disruption ofnucleic acid of or associated with the gene, such as at the DNA level.Exemplary methods for gene disruption include gene silencing, knockdown,knockout, and/or gene disruption techniques, such as gene editing.Examples include antisense technology, such as RNAi, siRNA, shRNA,and/or ribozymes, which generally result in transient reduction ofexpression, as well as gene editing techniques which result in targetedgene inactivation or disruption, e.g., by induction of breaks and/orhomologous recombination. Examples include insertions, mutations, anddeletions. The disruptions typically result in the repression and/orcomplete absence of expression of a normal or “wild type” productencoded by the gene. Exemplary of such gene disruptions are insertions,frameshift and mis sense mutations, deletions, knock-in, and knock-outof the gene or part of the gene, including deletions of the entire gene.Such disruptions can occur in the coding region, e.g., in one or moreexons, resulting in the inability to produce a full-length product,functional product, or any product, such as by insertion of a stopcodon. Such disruptions may also occur by disruptions in the promoter orenhancer or other region affecting activation of transcription, so as toprevent transcription of the gene. Gene disruptions include genetargeting, including targeted gene inactivation by homologousrecombination.

II. MULTIPLEX GENE EDITING

In certain embodiments, the present disclosure concerns multiplex geneediting of any type of immune cells. CRISPR is one example that can beused to disrupt the expression of two or more genes, such as 3, 4, 5, 6,7, 8, 9, 10, or more genes in an immune cell. The genes may be selectedfrom the genes listed in Table 1, such as NK Cell Receptor A (NKG2A),Sialic Acid-Binding Ig-Like Lectin 7 (SIGLEC-7, CD328), LymphocyteActivating 3 (LAG3), T-Cell Immunoglobulin Mucin Family Member 3 (TIM3,CD366, HAVCR2), Cytokine Inducible SH2-Containing Protein (CISH, CIS-1,SOCS), Forkhead Box O1 (FOXO1), Transforming Growth Factor Beta Receptor2 (TGFβR2), T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT),CD96, Adenosine Receptor 2A (ADORA2), Nuclear Receptor Subfamily 3 GroupC Member 1 (NR3C1), Programmed Cell Death 1 (PD1), Programmed Cell Death1 Ligand 1 (PDL-1), Programmed Cell Death 1 Ligand 2 (PDL-2), CD47,Signal Regulatory Protein Alpha (SIRPA), SH2 Domain-Containing Inositol5-Phosphatase 1 (SHIN), ADAM Metallopeptidase Domain 17 (ADAM17),Ribosomal Protein S6 (RPS6), Eukaryotic Translation Initiation Factor 4EBinding Protein 1 (4EBP1), CD25, CD40, Interleukin 21 Receptor (IL21R),Intercellular Adhesion Molecule 1 (ICAM1), CD95, CD80, CD86, Interleukin21 Receptor (IL10R), CD5, CD7, or there may be other inhibitory genes.The gene editing allows for simultaneous disruption of expression of themultiple genes.

In some embodiments, the gene disruption is carried out by effecting adisruption in the gene, such as a knock-out, insertion, missense orframeshift mutation, such as biallelic frameshift mutation, deletion ofall or part of the gene, e.g., one or more exons or portions therefore,and/or knock-in. For example, the disruption can be effected besequence-specific or targeted nucleases, including DNA-binding targetednucleases such as zinc finger nucleases (ZFN) and transcriptionactivator-like effector nucleases (TALENs), and RNA-guided nucleasessuch as a CRISPR-associated nuclease (Cas), specifically designed to betargeted to the sequence of the gene or a portion thereof.

In some embodiments, the disruption is transient or reversible, suchthat expression of the gene is restored at a later time. In otherembodiments, the disruption is not reversible or transient, e.g., ispermanent.

In some embodiments, gene disruption is carried out by induction of oneor more double-stranded breaks and/or one or more single-stranded breaksin the gene, typically in a targeted manner. In some embodiments, thedouble-stranded or single-stranded breaks are made by a nuclease, e.g.,an endonuclease, such as a gene-targeted nuclease. In some aspects, thebreaks are induced in the coding region of the gene, e.g., in an exon.For example, in some embodiments, the induction occurs near theN-terminal portion of the coding region, e.g., in the first exon, in thesecond exon, or in a subsequent exon.

The immune cell may be introduced to a guide RNA and CRISPR enzyme, ormRNA encoding the CRISPR enzyme. In some aspects, the cell is introducedto 1, 2, 3, 4, 5, or more guide RNAs simultaneously. For example, thecell may be introduced to 1, 2, or 3 guide RNAs during a firstelectroporation and then further introduced to 1, 2, or 3 additionalguide RNAs during a second electroporation, and so forth.

In some embodiments, gene disruption is achieved using antisensetechniques, such as by RNA interference (RNAi), short interfering RNA(siRNA), short hairpin (shRNA), and/or ribozymes are used to selectivelysuppress or repress expression of the gene. siRNA technology is RNAithat employs a double-stranded RNA molecule having a sequence homologouswith the nucleotide sequence of mRNA that is transcribed from the gene,and a sequence complementary with the nucleotide sequence. siRNAgenerally is homologous/complementary with one region of mRNA that istranscribed from the gene, or may be siRNA including a plurality of RNAmolecules that are homologous/complementary with different regions. Insome aspects, the siRNA is comprised in a polycistronic construct.

In some embodiments, the disruption is achieved using a DNA-targetingmolecule, such as a DNA-binding protein or DNA-binding nucleic acid, orcomplex, compound, or composition, containing the same, whichspecifically binds to or hybridizes to the gene. In some embodiments,the DNA-targeting molecule comprises a DNA-binding domain, e.g., a zincfinger protein (ZFP) DNA-binding domain, a transcription activator-likeprotein (TAL) or TAL effector (TALE) DNA-binding domain, a clusteredregularly interspaced short palindromic repeats (CRISPR) DNA-bindingdomain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE,and CRISPR system binding domains can be engineered to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Engineered DNA binding proteins(zinc fingers or TALEs) are proteins that are non-naturally occurring.Rational criteria for design include application of substitution rulesand computerized algorithms for processing information in a databasestoring information of existing ZFP and/or TALE designs and bindingdata.

For CRISPR-mediated disruption, the guide RNA and endonuclease may beintroduced to the immune cells by any means known in the art to allowdelivery inside cells or subcellular compartments, and agents/chemicalsand/or molecules (proteins and nucleic acids) that can be used includeliposomal delivery means, polymeric carriers, chemical carriers,lipoplexes, polyplexes, dendrimers, nanoparticles, emulsion, naturalendocytosis or phagocytose pathway as non-limiting examples, as well asphysical methods, such as electroporation. In specific aspects,electroporation is used to introduce the guide RNA and endonuclease, ornucleic acid encoding the endonuclease.

In one exemplary, specific method, the method for CRISPR knockout ofmultiple genes may comprise isolation of immune cells, such as NK cells,from cord blood or peripheral blood. The NK cells may be isolated andseeded on culture plates with irradiated feeder cells, such as at a 1:2ratio, as one example. The cells can then be electroporated with gRNAand Cas9 in the presence of IL-2, such as at a concentration of 200IU/mL. The media may be changed every other day, as one example. After1-3 days, the NK cells are isolated to remove the feeder cells and canthen be transduced with a CAR construct. The NK cells may then besubjected to a second CRISPR Cas9 knockout for additional gene(s). Afterthe electroporation, the NK cells may be seeded with feeder cells, suchas for 5-9 days.

TABLE 1 Genes for multiplex editing or CAR knock-in. Exemplary locationsfor knock-in are indicated. NK Cells, T cells, or MSC cells NKG2A Exon 4SIGLEC-7 Exon 1 LAG3 Exon 1 TIM3 Exon 2 CISH Exon 5 FOXO1 Exon 1 TGFBR2Exon 5 TIGIT Exon 2 CD96 Exon 2 ADORA2 Exon 2 NR3C1 Exon 2 PD1 Exon 1PDL-1 Exon 3 PDL-2 Exon 3 CD47 Exon 2 SIRPA Exon 2 SHIP1 Exon 1 ADAM17Exon 1 B2M Exon 2 CD16 B cells or T cells RPSS6 Exon 2 4EBP1 Exon 4 CD25Exon 3 CD40 Exon 3 IL21R Exon 1 ICAM1 Exon 4 CD95 Exon 2 CD80 Exon 3CD86 Exon 1 IL10R Exon 3 CD5 CD7 Exon 2

TABLE 2 Exemplary gRNA Sequences for Gene Knockout. CISH (Exon 4)AGGCCACATAGTGCTGCACA (gRNA1); SEQ ID NO: 1 TGTACAGCAGTGGCTGGTGG (gRNA2);SEQ ID NO: 2 NKG2A (Exon 4) AACAACTATCGTTACCACAG; SEQ ID NO: 3A2AR (Exon 3) CTCCTCGGTGTACATCACGG (gRNA1); SEQ ID NO: 4AGTAGTTGGTGACGTTCTGC (gRNA2); SEQ ID NO: 5 TIGIT (Exon 3)ACCCTGATGGGACGTACACT; SEQ ID NO: 6 CD96 (Exon 2) AGGCACAGTAGAAGCCGTAT;SEQ ID NO: 7 TIM3 (Exon 2) AGACGGGCACGAGGTTCCCT; SEQ ID NO: 8SHP1 (Exon 4) TCACGCACAAGAAACGTCCA; SEQ ID NO: 9 PD1 (Exon 2)CCCCTTCGGTCACCACGAGC; SEQ ID NO: 10 PDL1 (Exon 3) ATTTACTGTCACGGTTCCCA;SEQ ID NO: 11 PDL2 (Exon 3) CCCCATAGATGATTATGCAT; SEQ ID NO: 12TGFBR2 (Exon 5) GACGGCTGAGGAGCGGAAGA (gRNA1); SEQ ID NO: 13TGTGGAGGTGAGCAATCCCC (gRNA2); SEQ ID NO: 14

In some embodiments, the immune cells of the present disclosure aremodified to have altered expression of two or more genes. In someembodiments, the altered gene expression is carried out by effecting adisruption in the gene, such as a knock-out, insertion, missense orframeshift mutation, such as biallelic frameshift mutation, deletion ofall or part of the gene, e.g., one or more exon or portion therefore,and/or knock-in. In specific embodiments, the altered gene expressioncan be effected by sequence-specific or targeted nucleases, includingDNA-binding targeted nucleases such as RNA-guided nucleases such as aCRISPR-associated nuclease (Cas), specifically designed to be targetedto the sequence of the gene or a portion thereof.

In some embodiments, the alteration of the expression, activity, and/orfunction of the gene is carried out by disrupting the gene. In someaspects, the gene is modified so that its expression is reduced by atleast at or about 10, 20, 30, or 40%, generally at least at or about 50,60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% as comparedto the expression in the absence of the gene modification or in theabsence of the components introduced to effect the modification.

In some embodiments, the alteration is transient or reversible, suchthat expression of the gene is restored at a later time if desired. Inother embodiments, the alteration is not reversible or transient, e.g.,is permanent.

In some embodiments, gene alteration is carried out by induction of oneor more double-stranded breaks and/or one or more single-stranded breaksin the gene, typically in a targeted manner. In some embodiments, thedouble-stranded or single-stranded breaks are made by a nuclease, e.g.an endonuclease, such as a gene-targeted nuclease. In some aspects, thebreaks are induced in the coding region of the gene, e.g. in an exon.For example, in some embodiments, the induction occurs near theN-terminal portion of the coding region, e.g. in the first exon, in thesecond exon, or in a subsequent exon.

In some aspects, the double-stranded or single-stranded breaks undergorepair via a cellular repair process, such as by non-homologousend-joining (NHEJ) or homology-directed repair (HDR). In some aspects,the repair process is error-prone and results in disruption of the gene,such as a frameshift mutation, e.g., biallelic frameshift mutation,which can result in complete knockout of the gene. For example, in someaspects, the disruption comprises inducing a deletion, mutation, and/orinsertion. In some embodiments, the disruption results in the presenceof an early stop codon. In some aspects, the presence of an insertion,deletion, translocation, frameshift mutation, and/or a premature stopcodon results in disruption of the expression, activity, and/or functionof the gene.

In some embodiments, the alteration is carried out using one or moreDNA-binding nucleic acids, such as alteration via an RNA-guidedendonuclease (RGEN). For example, the alteration can be carried outusing clustered regularly interspaced short palindromic repeats (CRISPR)and CRISPR-associated (Cas) proteins. In general, “CRISPR system” referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g., tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), and/or other sequences andtranscripts from a CRISPR locus.

The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include anon-coding RNA molecule (guide) RNA, which sequence-specifically bindsto DNA, and a Cas protein (e.g., Cas9), with nuclease functionality(e.g., two nuclease domains). One or more elements of a CRISPR systemcan derive from a type I, type II, or type III CRISPR system, e.g.,derived from a particular organism comprising an endogenous CRISPRsystem, such as Streptococcus pyogenes.

In some aspects, a Cas nuclease and gRNA (including a fusion of crRNAspecific for the target sequence and fixed tracrRNA) are introduced intothe cell. In general, target sites at the 5′ end of the gRNA target theCas nuclease to the target site, e.g., the gene, using complementarybase pairing. The target site may be selected based on its locationimmediately 5′ of a protospacer adjacent motif (PAM) sequence, such astypically NGG, or NAG. In this respect, the gRNA is targeted to thedesired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14,12, 11, or 10 nucleotides of the guide RNA to correspond to the targetDNA sequence. In general, a CRISPR system is characterized by elementsthat promote the formation of a CRISPR complex at the site of a targetsequence. Typically, “target sequence” generally refers to a sequence towhich a guide sequence is designed to have complementarity, wherehybridization between the target sequence and a guide sequence promotesthe formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex.

The CRISPR system can induce double stranded breaks (DSBs) at the targetsite, followed by disruptions or alterations as discussed herein. Inother embodiments, Cas9 variants, deemed “nickases,” are used to nick asingle strand at the target site. Paired nickases can be used, e.g., toimprove specificity, each directed by a pair of different gRNAstargeting sequences such that upon introduction of the nickssimultaneously, a 5′ overhang is introduced. In other embodiments,catalytically inactive Cas9 is fused to a heterologous effector domainsuch as a transcriptional repressor or activator, to affect geneexpression.

The target sequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. The target sequence may be located in the nucleus orcytoplasm of the cell, such as within an organelle of the cell.Generally, a sequence or template that may be used for recombinationinto the targeted locus comprising the target sequences is referred toas an “editing template” or “editing polynucleotide” or “editingsequence”. In some aspects, an exogenous template polynucleotide may bereferred to as an editing template. In some aspects, the recombinationis homologous recombination.

Typically, in the context of an endogenous CRISPR system, formation ofthe CRISPR complex (comprising the guide sequence hybridized to thetarget sequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.The tracr sequence, which may comprise or consist of all or a portion ofa wild-type tracr sequence (e.g. about or more than about 20, 26, 32,45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracrsequence), may also form part of the CRISPR complex, such as byhybridization along at least a portion of the tracr sequence to all or aportion of a tracr mate sequence that is operably linked to the guidesequence. The tracr sequence has sufficient complementarity to a tracrmate sequence to hybridize and participate in formation of the CRISPRcomplex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% ofsequence complementarity along the length of the tracr mate sequencewhen optimally aligned.

One or more vectors driving expression of one or more elements of theCRISPR system can be introduced into the cell such that expression ofthe elements of the CRISPR system direct formation of the CRISPR complexat one or more target sites. Components can also be delivered to cellsas proteins and/or RNA. For example, a Cas enzyme, a guide sequencelinked to a tracr-mate sequence, and a tracr sequence could each beoperably linked to separate regulatory elements on separate vectors.Alternatively, two or more of the elements expressed from the same ordifferent regulatory elements, may be combined in a single vector, withone or more additional vectors providing any components of the CRISPRsystem not included in the first vector. The vector may comprise one ormore insertion sites, such as a restriction endonuclease recognitionsequence (also referred to as a “cloning site”). In some embodiments,one or more insertion sites are located upstream and/or downstream ofone or more sequence elements of one or more vectors. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell.

A vector may comprise a regulatory element operably linked to anenzyme-coding sequence encoding the CRISPR enzyme, such as a Casprotein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologs thereof, or modified versions thereof. Theseenzymes are known; for example, the amino acid sequence of S. pyogenesCas9 protein may be found in the SwissProt database under accessionnumber Q99ZW2.

The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia).The CRISPR enzyme can direct cleavage of one or both strands at thelocation of a target sequence, such as within the target sequence and/orwithin the complement of the target sequence. The vector can encode aCRISPR enzyme that is mutated with respect to a corresponding wild-typeenzyme such that the mutated CRISPR enzyme lacks the ability to cleaveone or both strands of a target polynucleotide containing a targetsequence. For example, an aspartate-to-alanine substitution (D10A) inthe RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 froma nuclease that cleaves both strands to a nickase (cleaves a singlestrand). In some embodiments, a Cas9 nickase may be used in combinationwith guide sequence(s), e.g., two guide sequences, which targetrespectively sense and antisense strands of the DNA target. Thiscombination allows both strands to be nicked and used to induce NHEJ orHDR.

In some embodiments, an enzyme coding sequence encoding the CRISPRenzyme is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon of the native sequence with codons that are morefrequently or most frequently used in the genes of that host cell whilemaintaining the native amino acid sequence. Various species exhibitparticular bias for certain codons of a particular amino acid. Codonbias (differences in codon usage between organisms) often correlateswith the efficiency of translation of messenger RNA (mRNA), which is inturn believed to be dependent on, among other things, the properties ofthe codons being translated and the availability of particular transferRNA (tRNA) molecules. The predominance of selected tRNAs in a cell isgenerally a reflection of the codons used most frequently in peptidesynthesis. Accordingly, genes can be tailored for optimal geneexpression in a given organism based on codon optimization.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof the CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97%, 99%, or more.

Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g. the Burrows WheelerAligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies,ELAND (IIlumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

The CRISPR enzyme may be part of a fusion protein comprising one or moreheterologous protein domains. A CRISPR enzyme fusion protein maycomprise any additional protein sequence, and optionally a linkersequence between any two domains. Examples of protein domains that maybe fused to a CRISPR enzyme include, without limitation, epitope tags,reporter gene sequences, and protein domains having one or more of thefollowing activities: methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity and nucleic acid binding activity. Non-limitingexamples of epitope tags include histidine (His) tags, V5 tags, FLAGtags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, andthioredoxin (Trx) tags. Examples of reporter genes include, but are notlimited to, glutathione-5-transferase (GST), horseradish peroxidase(HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US 20110059502, incorporated herein byreference.

III. INSERTION OF CAR AND/OR TCR AT INHIBITORY GENE LOCUS

In some embodiments, the present disclosure concerns the insertion ofCAR and/or TCR at a specific gene locus of an immune cells. The CARand/or TCR may be inserted at an inhibitory gene locus, such as a geneselected from the group consisting of NKG2A, Siglec 7, LAG3, TIM3, CISH,FOXO1, TGFBR2, TIGIT, CD96, Adenosine Receptor 2A, NR3C1, PD1, PDL-1,PDL-2, CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1,CD95, CD80, CD86, IL10R, CD5, CD7, and a combination thereof.

Inserting one or more CARs and/or TCRs in any of the methods disclosedherein can be site-specific. For example, one or more CARs and/or TCRscan be inserted adjacent to or near a promoter. In another example, oneor more transgenes can be inserted adjacent to, near, or within an exonof a gene (e.g., an inhibitory gene). Such insertions can be used toknock-in a CAR and/or TCR while simultaneously disrupting expression ofthe gene. In another example, one or more CARs and/or TCRs can beinserted adjacent to, near, or within an intron of a gene. A CAR and/orTCR can be introduced by an adeno-associated viral (AAV) viral vectorand integrate into a targeted genomic location. In some cases, a rAAVvector can be utilized to direct insertion of a transgene into a certainlocation. For example in some cases, a CAR and/or TCR can be integratedinto at least a portion of a NKG2A, Siglec 7, LAG3, TIM3, CISH, FOXO1,TGFBR2, TIGIT, CD96, Adenosine Receptor 2A, NR3C1, PD1, PDL-1, PDL-2,CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95,CD80, CD86, IL10R, CD5, or CD7 gene by a rAAV or an AAV vector.

Modification of a targeted locus of a cell can be produced byintroducing DNA into cells, where the DNA has homology to the targetlocus. DNA can include a marker gene, allowing for selection of cellscomprising the integrated construct. Complementary DNA in a targetvector can recombine with a chromosomal DNA at a target locus. A markergene can be flanked by complementary DNA sequences, a 3′ recombinationarm, and a 5′ recombination arm. Multiple loci within a cell can betargeted. For example, transgenes with recombination arms specific to 1or more target loci can be introduced at once such that multiple genomicmodifications occur in a single step. Homology arms can be about 0.2 kbto about 5 kb in length, such as from about 0.2 kb, 0.4 kb 0.6 kb, 0.8kb, 1.0 kb, 1.2 kb, 1.4 kb, 1.6 kb, 1.8 kb, 2.0 kb, 2.2 kb, 2.4 kb, 2.6kb, 2.8 kb, 3.0 kb, 3.2 kb, 3.4 kb, 3.6 kb, 3.8 kb, 4.0 kb, 4.2 kb, 4.4kb, 4.6 kb, 4.8 kb, to about 5.0 kb in length, for example.

In one method, guide RNA can be designed to target a region of aninhibitory gene locus, such as the adjacent to the promoter, an exon, orintron of the gene. The guide RNA may targeting the 5′ end of the anexon, such as the first, second, or third exon, of an inhibitory gene.The guide RNA may be comprise in an AAV vector repair matrix. The AAVvector may encode a self-cleaving 2A peptide, such as a P2A peptide,followed by the CAR cDNA. The CAR cassette and guide RNA sequence may beflanked by homology arms to the inhibitory gene. The immune cell maythen be introduced to, such as electroporated with, the AAV vector andCas9, such as Cas9 mRNA.

IV. IMMUNE CELLS

Certain embodiments of the present disclosure concern immune cells thatare engineered to have knockout of multiple genes and/or to haveknocking of a CAR at an inhibitory gene locus. The immune cells may be Tcells (e.g., regulatory T cells, CD4⁺ T cells, CD8⁺ T cells, orgamma-delta T cells), NK cells, invariant NK cells, NKT cells, B cells,stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotentstem (iPSC) cells). The immune cells may be virus-specific, express aCAR, and/or express a TCR. In some embodiments, the cells are monocytesor granulocytes, e.g., myeloid cells, macrophages, neutrophils,dendritic cells, mast cells, eosinophils, and/or basophils. Alsoprovided herein are methods of producing and engineering the immunecells as well as methods of using and administering the cells foradoptive cell therapy, in which case the cells may be autologous orallogeneic. Thus, the immune cells may be used as immunotherapy, such asto target cancer cells.

The immune cells may be isolated from subjects, particularly humansubjects. The immune cells can be obtained from a subject of interest,such as a subject suspected of having a particular disease or condition,a subject suspected of having a predisposition to a particular diseaseor condition, or a subject who is undergoing therapy for a particulardisease or condition. Immune cells can be collected from any location inwhich they reside in the subject including, but not limited to, blood,cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolatedimmune cells may be used directly, or they can be stored for a period oftime, such as by freezing.

The immune cells may be enriched/purified from any tissue where theyreside including, but not limited to, blood (including blood collectedby blood banks or cord blood banks), spleen, bone marrow, tissuesremoved and/or exposed during surgical procedures, and tissues obtainedvia biopsy procedures. Tissues/organs from which the immune cells areenriched, isolated, and/or purified may be isolated from both living andnon-living subjects, wherein the non-living subjects are organ donors.In particular embodiments, the immune cells are isolated from blood,such as peripheral blood or cord blood or a mixture thereof. In someaspects, immune cells isolated from cord blood have enhancedimmunomodulation capacity, such as measured by CD4-positive orCD8-positive T cell suppression. In specific aspects, the immune cellsare isolated from pooled blood, particularly pooled cord blood, forenhanced immunomodulation capacity. The pooled blood may be from 2 ormore sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g.,donor subjects).

The population of immune cells can be obtained from a subject in need oftherapy or suffering from a disease associated with reduced immune cellactivity. Thus, the cells may be autologous to the subject in need oftherapy. Alternatively, the population of immune cells can be obtainedfrom a donor, preferably a histocompatibility matched donor. The immunecell population can be harvested from the peripheral blood, cord blood,bone marrow, spleen, or any other organ/tissue in which immune cellsreside in the subject or donor. The immune cells can be isolated from apool of subjects and/or donors, such as from pooled cord blood.

When the population of immune cells is obtained from a donor distinctfrom the subject, the donor is preferably allogeneic, provided the cellsobtained are subject-compatible in that they can be introduced into thesubject. Allogeneic donor cells are may or may not behuman-leukocyte-antigen (HLA)-compatible.

A. T Cells

In some embodiments, the immune cells are T cells. Several basicapproaches for the derivation, activation and expansion of functionalanti-tumor effector cells have been described in the last two decades.These include: autologous cells, such as tumor-infiltrating lymphocytes(TILs); T cells activated ex-vivo using autologous DCs, lymphocytes,artificial antigen-presenting cells (APCs) or beads coated with T cellligands and activating antibodies, or cells isolated by virtue ofcapturing target cell membrane; allogeneic cells naturally expressinganti-host tumor T cell receptor (TCR); and non-tumor-specific autologousor allogeneic cells genetically reprogrammed or “redirected” to expresstumor-reactive TCR or chimeric TCR molecules displaying antibody-liketumor recognition capacity known as “T-bodies”. These approaches havegiven rise to numerous protocols for T cell preparation and immunizationwhich can be used in the methods described herein.

In some embodiments, the T cells are derived from the blood, bonemarrow, lymph, umbilical cord, or lymphoid organs. In some aspects, thecells are human cells. The cells typically are primary cells, such asthose isolated directly from a subject and/or isolated from a subjectand frozen. In some embodiments, the cells include one or more subsetsof T cells or other cell types, such as whole T cell populations, CD4⁺cells, CD8⁺ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. In some aspects, such asfor off-the-shelf technologies, the cells are pluripotent and/ormultipotent, such as stem cells, such as induced pluripotent stem cells(iPSCs). In some embodiments, the methods include isolating cells fromthe subject, preparing, processing, culturing, and/or engineering them,as described herein, and re-introducing them into the same patient,before or after cryopreservation.

Among the sub-types and subpopulations of T cells (e.g., CD4⁺ and/orCD8⁺ T cells) are naive T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(TSC_(M)), central memory T (TC_(M)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, one or more of the T cell populations is enrichedfor or depleted of cells that are positive for a specific marker, suchas surface markers, or that are negative for a specific marker. In somecases, such markers are those that are absent or expressed at relativelylow levels on certain populations of T cells (e.g., non-memory cells)but are present or expressed at relatively higher levels on certainother populations of T cells (e.g., memory cells).

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8⁺ T cells are further enriched for or depletedof naive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations.

In some embodiments, the T cells are autologous T cells. In this method,tumor samples are obtained from patients and a single cell suspension isobtained. The single cell suspension can be obtained in any suitablemanner, e.g., mechanically (disaggregating the tumor using, e.g., agentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) orenzymatically (e.g., collagenase or DNase). Single-cell suspensions oftumor enzymatic digests are cultured in interleukin-2 (IL-2).

The cultured T cells can be pooled and rapidly expanded. Rapid expansionprovides an increase in the number of antigen-specific T-cells of atleast about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, orgreater) over a period of about 10 to about 14 days. More preferably,rapid expansion provides an increase of at least about 200-fold (e.g.,200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over aperiod of about 10 to about 14 days.

Expansion can be accomplished by any of a number of methods as are knownin the art. For example, T cells can be rapidly expanded usingnon-specific T-cell receptor stimulation in the presence of feederlymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15),with IL-2 being preferred. The non-specific T-cell receptor stimulus caninclude around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cellscan be rapidly expanded by stimulation of peripheral blood mononuclearcells (PBMC) in vitro with one or more antigens (including antigenicportions thereof, such as epitope(s), or a cell) of the cancer, whichcan be optionally expressed from a vector, such as an human leukocyteantigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growthfactor, such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred. Thein vitro-induced T-cells are rapidly expanded by re-stimulation with thesame antigen(s) of the cancer pulsed onto HLA-A2-expressingantigen-presenting cells. Alternatively, the T-cells can bere-stimulated with irradiated, autologous lymphocytes or with irradiatedHLA-A2⁺ allogeneic lymphocytes and IL-2, for example.

The autologous T cells can be modified to express a T cell growth factorthat promotes the growth and activation of the autologous T cells.Suitable T cell growth factors include, for example, interleukin (IL)-2,IL-7, IL-15, and IL-12. Suitable methods of modification are known inthe art. See, for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 2001; and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994.In particular aspects, modified autologous T cells express the T cellgrowth factor at high levels. T cell growth factor coding sequences,such as that of IL-12, are readily available in the art, as arepromoters, the operable linkage of which to a T cell growth factorcoding sequence promote high-level expression.

B. NK Cells

In some embodiments, the immune cells are natural killer (NK) cells. NKcells are a subpopulation of lymphocytes that have spontaneouscytotoxicity against a variety of tumor cells, virus-infected cells, andsome normal cells in the bone marrow and thymus. NK cells differentiateand mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus.NK cells can be detected by specific surface markers, such as CD16,CD56, and CD8 in humans. NK cells do not express T cell antigenreceptors, the pan T marker CD3, or surface immunoglobulin B cellreceptors.

In certain embodiments, NK cells are derived from human peripheral bloodmononuclear cells (PBMC), unstimulated leukapheresis products (PBSC),human embryonic stem cells (hESCs), induced pluripotent stem cells(iPSCs), bone marrow, or umbilical cord blood by methods well known inthe art. Particularly, umbilical CB is used to derive NK cells. Incertain aspects, the NK cells are isolated and expanded by thepreviously described method of ex vivo expansion of NK cells (Spanholtzet al., 2011; Shah et al., 2013). In this method, CB mononuclear cellsare isolated by ficoll density gradient centrifugation and cultured in abioreactor with IL-2 and artificial antigen presenting cells (aAPCs).After 7 days, the cell culture is depleted of any cells expressing CD3and re-cultured for an additional 7 days. The cells are againCD3-depleted and characterized to determine the percentage of CD56⁺/CD3⁻cells or NK cells. In other methods, umbilical CB is used to derive NKcells by the isolation of CD34⁺ cells and differentiation intoCD56⁺/CD3⁻ cells by culturing in medium contain SCF, IL-7, IL-15, andIL-2.

In specific embodiments, the NK cells are expanded at some point duringtheir preparation. In specific cases, expansion of the NK cellscomprises: stimulating mononuclear cells (MNCs) from cord blood in thepresence of antigen presenting cells (APCs) and IL-2; and re-stimulatingthe cells with APCs to produce expanded NK cells, wherein in at leastsome cases the method is performed in a bioreactor. The stimulating stepcan direct the MNCs towards NK cells. The re-stimulating step may or maynot comprise the presence of IL-2. In particular aspects, the methoddoes not comprise removal or addition of any media components during astimulating step. In particular aspects, the method is performed withina certain time frame, such as in less than 15 days, for example in 14days.

In a certain embodiment, the NK cells are expanded by an ex vivo methodfor the expansion comprising: (a) obtaining a starting population ofmononuclear cells (MNCs) from cord blood; (b) stimulating the MNCs inthe presence of antigen presenting cells (APCs) and IL-2; and (c)re-stimulating the cells with APCs to produce expanded NK cells, whereinthe method is performed in a bioreactor and is good manufacturingpractice (GMP) compliant. The stimulating of step (b) can direct theMNCs towards NK cells. Step (c) may or may not comprise the presence ofIL-2. In particular aspects, the method does not comprise removal oraddition of any media components during step (b). In particular aspects,the method is performed in less than 15 days, such as in 14 days.

In some aspects, the method further comprises depleting cells positivefor one or more particular markers, such as CD3, for example. In certainaspects, the depleting step is performed between steps (b) and (c). Insome aspects, the cells are removed from the bioreactor for CD3depletion and placed in the bioreactor for step (c).

In certain aspects, obtaining the starting population of MNCs from cordblood comprises thawing cord blood in the presence of dextran, humanserum albumin (HSA), DNAse, and/or magnesium chloride. In particularaspects, obtaining the starting population of MNCs from cord bloodcomprises thawing cord blood in the presence of dextran and/or DNase. Inspecific aspects, the cord blood is washed in the presence of 5-20%,such as 10%, dextran. In certain aspects, the cord blood is suspended inthe presence of magnesium chloride, such as at a concentration of100-300 mM, particularly 200 mM. In some aspects, obtaining comprisesperforming ficoll density gradient centrifugation to obtain mononuclearcells (MNCs).

In certain aspects, the bioreactor is a gas permeable bioreactor. Inparticular aspects, the gas permeable bioreactor is G-Rex100M orG-Rex100. In some aspects, the stimulating of step (b) is performed in3-5 L of media, such as 3, 3.5, 4, 4.5, or 5 L.

In some aspects, the APCs are gamma-irradiated. In certain aspects, theAPCs are engineered to express membrane-bound IL-21 (mbIL-21). Inparticular aspects, the APCs are engineered to express IL-21, IL-15,and/or IL-2. In some aspects, the MNCs and APCs are cultured at a ratioof 1:2. In some aspects, the IL-2 is at a concentration of 50-200 IU/mL,such as 100 IU/mL. In particular aspects, the IL-2 is replenished every2-3 days.

In particular aspects, step (b) is performed for 6-8 days, such as 7days. In some aspects, step (c) is performed for 6-8 days, such as 7days. In some aspects, step (c) does not comprise splitting of thecells. In particular aspects, the cells are fed twice with IL-2 duringstep (c), and in specific cases, no other media components are added orremoved during step (c).

In some aspects, the method comprises the use of 3, 4, 5, or 6bioreactors. In particular aspects, the method comprises the use of lessthan 10 bioreactors.

In specific aspects, the NK cells are expanded at least 500-fold,800-fold, 1000-fold, 1200-fold, 1500-fold, 2000-fold, 2500-fold,3000-fold, or 5000-fold. In particular aspects, culturing the NK cellsin the bioreactor produces more than 1000-fold NK cells as compared tostatic liquid culture.

In certain aspects, the method does not comprise human leukocyte antigen(HLA) matching. In some aspects, the starting population of NK cells arenot obtained from a haploidentical donor.

In some aspects, the expanded NK cells have enhanced anti-tumor activityas comprises to NK cells expanded from peripheral blood. In certainaspects, the expanded NK cells have higher expression of one or morecell cycle genes, one or more cell division genes, and/or one or moreDNA replication genes, as compared to NK cells expanded from peripheralblood. In some aspects, the expanded NK cells have higher proliferativecapacity as compared to NK cells expanded from peripheral blood. In someaspects, the expanded NK cells do not exhibit exhaustion. In certainaspects, exhaustion is detected by measuring expression of perforin,granzyme, CD57, KLRG1, and/or PD1. In some aspects, the expanded NKcells have high expression of perforin and/or granzyme. In certainaspects, the expanded NK cells have low or no expression of CD57, KLRG1,and/or PD1.

In some aspects, the expanded NK cells comprise a clinically relevantdose. In certain aspects, the cord blood is frozen cord blood. Inparticular aspects, the frozen cord blood has been tested for one ormore infectious diseases, such as hepatitis A, hepatitis B, hepatitis C,Trypanosoma cruzi, HIV, Human T-Lymphotropic virus, syphyllis, Zikavirus, and so forth. In some aspects, the cord blood is pooled cordblood, such as from 3, 4, 5, 6, 7, or 8 individual cord blood units.

In some aspects, the NK cells are not autologous, such as with respectto a recipient individual. In certain aspects, the NK cells are notallogeneic, such as with respect to a recipient individual.

In some aspects, the APCs are universal antigen presenting cells(uAPCs). In certain aspects, the uAPCs are engineered to express (1)CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21),and (3) 41BB ligand (41BBL). In some aspects, the uAPCs express CD48. Incertain aspects, the uAPCs express CS1. In particular aspects, the uAPCsexpress CD48 and CS1. In some aspects, the uAPCs have essentially noexpression of endogenous HLA class I, II, and/or CD1d molecules. Incertain aspects, the uAPCs express ICAM-1 (CD54) and/or LFA-3 (CD58). Inparticular aspects, the uAPCs are further defined as leukemiacell-derived aAPCs, such as K562 cells.

C. Stem Cells

In some embodiments, the immune cells of the present disclosure may bestem cells, such as induced pluripotent stem cells (PSCs), mesenchymalstem cells (MSCs), or hematopoietic stem cells (HSCs).

The pluripotent stem cells used herein may be induced pluripotent stem(iPS) cells, commonly abbreviated iPS cells or iPSCs. With the exceptionof germ cells, any cell can be used as a starting point for iPSCs. Forexample, cell types could be keratinocytes, fibroblasts, hematopoieticcells, mesenchymal cells, liver cells, or stomach cells. There is nolimitation on the degree of cell differentiation or the age of an animalfrom which cells are collected; even undifferentiated progenitor cells(including somatic stem cells) and finally differentiated mature cellscan be used as sources of somatic cells in the methods disclosed herein.

Somatic cells can be reprogrammed to produce iPS cells using methodsknown to one of skill in the art. Generally, nuclear reprogrammingfactors are used to produce pluripotent stem cells from a somatic cell.In some embodiments, at least three, or at least four, of Klf4, c-Myc,Oct3/4, Sox2, Nanog, and Lin28 are utilized. In other embodiments,Oct3/4, Sox2, c-Myc and Klf4 are utilized or Oct3/4, Sox2, Nanog, andLin28.

Once derived, iPSCs can be cultured in a medium sufficient to maintainpluripotency. In certain embodiments, undefined conditions may be used;for example, pluripotent cells may be cultured on fibroblast feedercells or a medium that has been exposed to fibroblast feeder cells inorder to maintain the stem cells in an undifferentiated state. In someembodiments, the cell is cultured in the co-presence of mouse embryonicfibroblasts treated with radiation or an antibiotic to terminate thecell division, as feeder cells. Alternately, pluripotent cells may becultured and maintained in an essentially undifferentiated state using adefined, feeder-independent culture system, such as a TESR™ medium orE8™/Essential 8™ medium.

V. GENETICALLY ENGINEERED ANTIGEN RECEPTORS

The immune cells of the present disclosure can be genetically engineeredto express antigen receptors such as engineered TCRs, CARs, chimericcytokine receptors, chemokine receptors, a combination thereof, and soon. For example, the immune cells are modified to express a CAR and/orTCR having antigenic specificity for a cancer antigen. Multiple CARsand/or TCRs, such as to different antigens, may be added to the immunecells. In some aspects, the immune cells are engineered to express theCAR or TCR by knock-in of the CAR or TCR at an inhibitory gene locususing CRISPR.

Suitable methods of modification are known in the art. See, forinstance, Sambrook and Ausubel, supra. For example, the cells may betransduced to express a TCR having antigenic specificity for a cancerantigen using transduction techniques described in Heemskerk et al.,2008 and Johnson et al., 2009.

Electroporation of RNA coding for the full length TCR α and β (or γ andδ) chains can be used as alternative to overcome long-term problems withautoreactivity caused by pairing of retrovirally transduced andendogenous TCR chains. Even if such alternative pairing takes place inthe transient transfection strategy, the possibly generated autoreactiveT cells will lose this autoreactivity after some time, because theintroduced TCR α and β chain are only transiently expressed. When theintroduced TCR α and β chain expression is diminished, only normalautologous T cells are left. This is not the case when full length TCRchains are introduced by stable retroviral transduction, which willnever lose the introduced TCR chains, causing a constantly presentautoreactivity in the patient.

In some embodiments, the cells comprise one or more nucleic acidsintroduced via genetic engineering that encode one or more antigenreceptors, and genetically engineered products of such nucleic acids. Insome embodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature (e.g.,chimeric).

In some embodiments, the CAR contains an extracellularantigen-recognition domain that specifically binds to an antigen. Insome embodiments, the antigen is a protein expressed on the surface ofcells. In some embodiments, the CAR is a TCR-like CAR and the antigen isa processed peptide antigen, such as a peptide antigen of anintracellular protein, which, like a TCR, is recognized on the cellsurface in the context of a major histocompatibility complex (MHC)molecule.

Exemplary antigen receptors, including CARs and recombinant TCRs, aswell as methods for engineering and introducing the receptors intocells, include those described, for example, in international patentapplication publication numbers WO200014257, WO2013126726,WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061U.S. patent application publication numbers US2002131960, US2013287748,US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592,8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209,7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patentapplication number EP2537416, and/or those described by Sadelain et al.,2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In someaspects, the genetically engineered antigen receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No.: WO/2014055668 A1.

A. Chimeric Antigen Receptors

In some embodiments, the CAR comprises: a) one or more intracellularsignaling domains, b) a transmembrane domain, and c) an extracellulardomain comprising an antigen binding region.

In some embodiments, the engineered antigen receptors include CARs,including activating or stimulatory CARs, costimulatory CARs (seeWO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al.,2013). The CARs generally include an extracellular antigen (or ligand)binding domain linked to one or more intracellular signaling components,in some aspects via linkers and/or transmembrane domain(s). Suchmolecules typically mimic or approximate a signal through a naturalantigen receptor, a signal through such a receptor in combination with acostimulatory receptor, and/or a signal through a costimulatory receptoralone.

Certain embodiments of the present disclosure concern the use of nucleicacids, including nucleic acids encoding an antigen-specific CARpolypeptide, including a CAR that has been humanized to reduceimmunogenicity (hCAR), comprising an intracellular signaling domain, atransmembrane domain, and an extracellular domain comprising one or moresignaling motifs. In certain embodiments, the CAR may recognize anepitope comprising the shared space between one or more antigens. Incertain embodiments, the binding region can comprise complementarydetermining regions of a monoclonal antibody, variable regions of amonoclonal antibody, and/or antigen binding fragments thereof. Inanother embodiment, that specificity is derived from a peptide (e.g.,cytokine) that binds to a receptor.

It is contemplated that the human CAR nucleic acids may be human genesused to enhance cellular immunotherapy for human patients. In a specificembodiment, the invention includes a full-length CAR cDNA or codingregion. The antigen binding regions or domain can comprise a fragment ofthe VH and VL chains of a single-chain variable fragment (scFv) derivedfrom a particular human monoclonal antibody, such as those described inU.S. Pat. No. 7,109,304, incorporated herein by reference. The fragmentcan also be any number of different antigen binding domains of a humanantigen-specific antibody. In a more specific embodiment, the fragmentis an antigen-specific scFv encoded by a sequence that is optimized forhuman codon usage for expression in human cells.

The arrangement could be multimeric, such as a diabody or multimers. Themultimers are most likely formed by cross pairing of the variableportion of the light and heavy chains into a diabody. The hinge portionof the construct can have multiple alternatives from being totallydeleted, to having the first cysteine maintained, to a proline ratherthan a serine substitution, to being truncated up to the first cysteine.The Fc portion can be deleted. Any protein that is stable and/ordimerizes can serve this purpose. One could use just one of the Fcdomains, e.g., either the CH2 or CH3 domain from human immunoglobulin.One could also use the hinge, CH2 and CH3 region of a humanimmunoglobulin that has been modified to improve dimerization. One couldalso use just the hinge portion of an immunoglobulin. One could also useportions of CD8alpha.

In some embodiments, the CAR nucleic acid comprises a sequence encodingother costimulatory receptors, such as a transmembrane domain and amodified CD28 intracellular signaling domain. Other costimulatoryreceptors include, but are not limited to one or more of CD28, CD27,OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to a primarysignal initiated by CD3 t, an additional signal provided by a humancostimulatory receptor inserted in a human CAR is important for fullactivation of NK cells and could help improve in vivo persistence andthe therapeutic success of the adoptive immunotherapy.

In some embodiments, CAR is constructed with a specificity for aparticular antigen (or marker or ligand), such as an antigen expressedin a particular cell type to be targeted by adoptive therapy, e.g., acancer marker, and/or an antigen intended to induce a dampeningresponse, such as an antigen expressed on a normal or non-diseased celltype. Thus, the CAR typically includes in its extracellular portion oneor more antigen binding molecules, such as one or more antigen-bindingfragment, domain, or portion, or one or more antibody variable domains,and/or antibody molecules. In some embodiments, the CAR includes anantigen-binding portion or portions of an antibody molecule, such as asingle-chain antibody fragment (scFv) derived from the variable heavy(VH) and variable light (VL) chains of a monoclonal antibody (mAb).

In certain embodiments of the chimeric antigen receptor, theantigen-specific portion of the receptor (which may be referred to as anextracellular domain comprising an antigen binding region) comprises atumor associated antigen or a pathogen-specific antigen binding domain.Antigens include carbohydrate antigens recognized by pattern-recognitionreceptors, such as Dectin-1. A tumor associated antigen may be of anykind so long as it is expressed on the cell surface of tumor cells.Exemplary embodiments of tumor associated antigens include CD19, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR,c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associatedantigen, mutated p53, mutated ras, and so forth. In certain embodiments,the CAR may be co-expressed with a cytokine to improve persistence whenthere is a low amount of tumor-associated antigen. For example, CAR maybe co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15,IL-12, IL-18, IL-21, or a combination thereof.

The sequence of the open reading frame encoding the chimeric receptorcan be obtained from a genomic DNA source, a cDNA source, or can besynthesized (e.g., via PCR), or combinations thereof. Depending upon thesize of the genomic DNA and the number of introns, it may be desirableto use cDNA or a combination thereof as it is found that intronsstabilize the mRNA. Also, it may be further advantageous to useendogenous or exogenous non-coding regions to stabilize the mRNA.

It is contemplated that the chimeric construct can be introduced intoimmune cells as naked DNA or in a suitable vector. Methods of stablytransfecting cells by electroporation using naked DNA are known in theart. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers tothe DNA encoding a chimeric receptor contained in a plasmid expressionvector in proper orientation for expression.

Alternatively, a viral vector (e.g., a retroviral vector, adenoviralvector, adeno-associated viral vector, or lentiviral vector) can be usedto introduce the chimeric construct into immune cells. Suitable vectorsfor use in accordance with the method of the present disclosure arenon-replicating in the immune cells. A large number of vectors are knownthat are based on viruses, where the copy number of the virus maintainedin the cell is low enough to maintain the viability of the cell, suchas, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

In some aspects, the antigen-specific binding, or recognition componentis linked to one or more transmembrane and intracellular signalingdomains. In some embodiments, the CAR includes a transmembrane domainfused to the extracellular domain of the CAR. In one embodiment, thetransmembrane domain that naturally is associated with one of thedomains in the CAR is used. In some instances, the transmembrane domainis selected or modified by amino acid substitution to avoid binding ofsuch domains to the transmembrane domains of the same or differentsurface membrane proteins to minimize interactions with other members ofthe receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T− cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37,CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D,and DAP molecules.

Alternatively the transmembrane domain in some embodiments is synthetic.In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain.

In certain embodiments, the platform technologies disclosed herein togenetically modify immune cells, such as NK cells, comprise (i)non-viral gene transfer using an electroporation device (e.g., anucleofector), (ii) CARs that signal through endodomains (e.g.,CD28/CD3-ζ CD137/CD3-ζ, or other combinations), (iii) CARs with variablelengths of extracellular domains connecting the antigen-recognitiondomain to the cell surface, and, in some cases, (iv) artificial antigenpresenting cells (aAPC) derived from K562 to be able to robustly andnumerically expand CAR⁺ immune cells (Singh et al., 2008; Singh et al.,2011).

B. T Cell Receptor (TCR)

In some embodiments, the genetically engineered antigen receptorsinclude recombinant TCRs and/or TCRs cloned from naturally occurring Tcells. A “T cell receptor” or “TCR” refers to a molecule that contains avariable α and β chains (also known as TCRα and TCRβ, respectively) or avariable γ and δ chains (also known as TCRγ and TCRδ, respectively) andthat is capable of specifically binding to an antigen peptide bound to aMHC receptor. In some embodiments, the TCR is in the αβ form.

Typically, TCRs that exist in αβ and γδ forms are generally structurallysimilar, but T cells expressing them may have distinct anatomicallocations or functions. A TCR can be found on the surface of a cell orin soluble form. Generally, a TCR is found on the surface of T cells (orT lymphocytes) where it is generally responsible for recognizingantigens bound to major histocompatibility complex (MHC) molecules. Insome embodiments, a TCR also can contain a constant domain, atransmembrane domain and/or a short cytoplasmic tail (see, e.g., Janewayet al, 1997). For example, in some aspects, each chain of the TCR canpossess one N-terminal immunoglobulin variable domain, oneimmunoglobulin constant domain, a transmembrane region, and a shortcytoplasmic tail at the C-terminal end. In some embodiments, a TCR isassociated with invariant proteins of the CD3 complex involved inmediating signal transduction. Unless otherwise stated, the term “TCR”should be understood to encompass functional TCR fragments thereof. Theterm also encompasses intact or full-length TCRs, including TCRs in theαβ form or γδ form.

Thus, for purposes herein, reference to a TCR includes any TCR orfunctional fragment, such as an antigen-binding portion of a TCR thatbinds to a specific antigenic peptide bound in an MHC molecule, i.e.MHC-peptide complex. An “antigen-binding portion” or antigen-bindingfragment” of a TCR, which can be used interchangeably, refers to amolecule that contains a portion of the structural domains of a TCR, butthat binds the antigen (e.g. MHC-peptide complex) to which the full TCRbinds. In some cases, an antigen-binding portion contains the variabledomains of a TCR, such as variable a chain and variable β chain of aTCR, sufficient to form a binding site for binding to a specificMHC-peptide complex, such as generally where each chain contains threecomplementarity determining regions.

In some embodiments, the variable domains of the TCR chains associate toform loops, or complementarity determining regions (CDRs) analogous toimmunoglobulins, which confer antigen recognition and determine peptidespecificity by forming the binding site of the TCR molecule anddetermine peptide specificity. Typically, like immunoglobulins, the CDRsare separated by framework regions (FRs) (see, e.g., Jores et al., 1990;Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3is the main CDR responsible for recognizing processed antigen, althoughCDR1 of the alpha chain has also been shown to interact with theN-terminal part of the antigenic peptide, whereas CDR1 of the beta chaininteracts with the C-terminal part of the peptide. CDR2 is thought torecognize the MHC molecule. In some embodiments, the variable region ofthe β-chain can contain a further hypervariability (HV4) region.

In some embodiments, the TCR chains contain a constant domain. Forexample, like immunoglobulins, the extracellular portion of TCR chains(e.g., α-chain, β-chain) can contain two immunoglobulin domains, avariable domain (e.g., V_(a) or Vp; typically amino acids 1 to 116 basedon Kabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5^(th) ed.) at the N-terminus, andone constant domain (e.g., a-chain constant domain or C_(a), typicallyamino acids 117 to 259 based on Kabat, β-chain constant domain or Cp,typically amino acids 117 to 295 based on Kabat) adjacent to the cellmembrane. For example, in some cases, the extracellular portion of theTCR formed by the two chains contains two membrane-proximal constantdomains, and two membrane-distal variable domains containing CDRs. Theconstant domain of the TCR domain contains short connecting sequences inwhich a cysteine residue forms a disulfide bond, making a link betweenthe two chains. In some embodiments, a TCR may have an additionalcysteine residue in each of the a and β chains such that the TCRcontains two disulfide bonds in the constant domains.

In some embodiments, the TCR chains can contain a transmembrane domain.In some embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chains contains a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3.For example, a TCR containing constant domains with a transmembraneregion can anchor the protein in the cell membrane and associate withinvariant subunits of the CD3 signaling apparatus or complex.

Generally, CD3 is a multi-protein complex that can possess threedistinct chains (γ, δ, and ε) in mammals and the ζ-chain. For example,in mammals the complex can contain a CD3γ chain, a CD3δ chain, two CD3εchains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, and CD3ε chainsare highly related cell surface proteins of the immunoglobulinsuperfamily containing a single immunoglobulin domain. The transmembraneregions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, whichis a characteristic that allows these chains to associate with thepositively charged T cell receptor chains. The intracellular tails ofthe CD3γ, CD3δ, and CD3ε chains each contain a single conserved motifknown as an immunoreceptor tyrosine-based activation motif or ITAM,whereas each CD3ζ chain has three. Generally, ITAMs are involved in thesignaling capacity of the TCR complex. These accessory molecules havenegatively charged transmembrane regions and play a role in propagatingthe signal from the TCR into the cell. The CD3- and ζ-chains, togetherwith the TCR, form what is known as the T cell receptor complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (α and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds. In some embodiments, a TCR for atarget antigen (e.g., a cancer antigen) is identified and introducedinto the cells. In some embodiments, nucleic acid encoding the TCR canbe obtained from a variety of sources, such as by polymerase chainreaction (PCR) amplification of publicly available TCR DNA sequences. Insome embodiments, the TCR is obtained from a biological source, such asfrom cells such as from a T cell (e.g. cytotoxic T cell), T cellhybridomas or other publicly available source. In some embodiments, theT cells can be obtained from in vivo isolated cells. In someembodiments, a high-affinity T cell clone can be isolated from apatient, and the TCR isolated. In some embodiments, the T cells can be acultured T cell hybridoma or clone. In some embodiments, the TCR clonefor a target antigen has been generated in transgenic mice engineeredwith human immune system genes (e.g., the human leukocyte antigensystem, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al.,2009 and Cohen et al., 2005). In some embodiments, phage display is usedto isolate TCRs against a target antigen (see, e.g., Varela-Rohena etal., 2008 and Li, 2005). In some embodiments, the TCR or antigen-bindingportion thereof can be synthetically generated from knowledge of thesequence of the TCR.

C. Antigen-Presenting Cells

Antigen-presenting cells, which include macrophages, B lymphocytes, anddendritic cells, are distinguished by their expression of a particularMHC molecule. APCs internalize antigen and re-express a part of thatantigen, together with the MHC molecule on their outer cell membrane.The MHC is a large genetic complex with multiple loci. The MHC lociencode two major classes of MHC membrane molecules, referred to as classI and class II MHCs. T helper lymphocytes generally recognize antigenassociated with MHC class II molecules, and T cytotoxic lymphocytesrecognize antigen associated with MHC class I molecules. In humans theMHC is referred to as the HLA complex and in mice the H-2 complex.

In some cases, aAPCs are useful in preparing therapeutic compositionsand cell therapy products of the embodiments. For general guidanceregarding the preparation and use of antigen-presenting systems, see,e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S.Patent Application Publication Nos. 2009/0017000 and 2009/0004142; andInternational Publication No. WO2007/103009.

aAPC systems may comprise at least one exogenous assisting molecule. Anysuitable number and combination of assisting molecules may be employed.The assisting molecule may be selected from assisting molecules such asco-stimulatory molecules and adhesion molecules. Exemplaryco-stimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, andIL-21. Adhesion molecules may include carbohydrate-binding glycoproteinssuch as selectins, transmembrane binding glycoproteins such asintegrins, calcium-dependent proteins such as cadherins, and single-passtransmembrane immunoglobulin (Ig) superfamily proteins, such asintercellular adhesion molecules (ICAMs), which promote, for example,cell-to-cell or cell-to-matrix contact. Exemplary adhesion moleculesinclude LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, andreagents useful for selection, cloning, preparation, and expression ofexemplary assisting molecules, including co-stimulatory molecules andadhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042,6,355,479, and 6,362,001.

D. Antigens

Among the antigens targeted by the genetically engineered antigenreceptors are those expressed in the context of a disease, condition, orcell type to be targeted via the adoptive cell therapy. Among thediseases and conditions are proliferative, neoplastic, and malignantdiseases and disorders, including cancers and tumors, includinghematologic cancers, cancers of the immune system, such as lymphomas,leukemias, and/or myelomas, such as B, T, and myeloid leukemias,lymphomas, and multiple myelomas. In some embodiments, the antigen isselectively expressed or overexpressed on cells of the disease orcondition, e.g., the tumor or pathogenic cells, as compared to normal ornon-targeted cells or tissues. In other embodiments, the antigen isexpressed on normal cells and/or is expressed on the engineered cells.

Any suitable antigen may be targeted in the present method. The antigenmay be associated with certain cancer cells but not associated withnon-cancerous cells, in some cases. Exemplary antigens include, but arenot limited to, antigenic molecules from infectious agents,auto-/self-antigens, tumor-/cancer-associated antigens, and tumorneoantigens (Linnemann et al., 2015). In particular aspects, theantigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3,Mage-A4, Mage-A10, TRAIL/DR4, and CEA. In particular aspects, theantigens for the two or more antigen receptors include, but are notlimited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2,CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4, and/or CEA. Thesequences for these antigens are known in the art, for example, in theGenBank® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No.NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No.NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (AccessionNo. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No.NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No.NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (AccessionNo. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL/DR4(Accession No. NC_000003.12), and/or CEA (Accession No. NC_000019.10).

Tumor-associated antigens may be derived from prostate, breast,colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver,brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder,thyroid, or melanoma cancers, as examples. Exemplary tumor-associatedantigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4(or other MAGE antigens such as those disclosed in International PatentPublication No. WO 99/40188); PRAME; BAGE; RAGE, Lage (also known as NYESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumorantigens are expressed in a wide range of tumor types such as melanoma,lung carcinoma, sarcoma, and bladder carcinoma. See, e.g., U.S. Pat. No.6,544,518. Prostate cancer tumor-associated antigens include, forexample, prostate specific membrane antigen (PSMA), prostate-specificantigen (PSA), prostatic acid phosphates, NKX3.1, and six-transmembraneepithelial antigen of the prostate (STEAP).

Other tumor associated antigens include Plu-1, HASH-1, HasH-2, Criptoand Criptin. Additionally, a tumor antigen may be a self-peptidehormone, such as whole length gonadotrophin hormone releasing hormone(GnRH), a short 10 amino acid long peptide, useful in the treatment ofmany cancers.

Tumor antigens include tumor antigens derived from cancers that arecharacterized by tumor-associated antigen expression, such as HER-2/neuexpression. Tumor-associated antigens of interest includelineage-specific tumor antigens such as the melanocyte-melanoma lineageantigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase andtyrosinase-related protein. Illustrative tumor-associated antigensinclude, but are not limited to, tumor antigens derived from orcomprising any one or more of, p53, Ras, c-Myc, cytoplasmicserine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf,cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3,-4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, Tyrosinase,TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2,Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2,SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin/m,Caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2,KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2,707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferonregulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR,Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptortyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (inparticular, EGFRvIII), platelet derived growth factor receptor (PDGFR),vascular endothelial growth factor receptor (VEGFR)), cytoplasmictyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linkedkinase (ILK), signal transducers and activators of transcription STAT3,STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2),Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch1-4), c-Met,mammalian targets of rapamycin (mTOR), WNT, extracellularsignal-regulated kinases (ERKs), and their regulatory subunits, PMSA,PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonicanhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1,GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2,ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgenreceptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1,mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS,SART3, STn, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2,XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fosrelated antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A,MAD2L1, CTAG1B, SUNC1, LRRN1 and idiotype.

Antigens may include epitopic regions or epitopic peptides derived fromgenes mutated in tumor cells or from genes transcribed at differentlevels in tumor cells compared to normal cells, such as telomeraseenzyme, survivin, mesothelin, mutated ras, bcr/abl rearrangement,Her2/neu, mutated or wild-type p53, cytochrome P450 1B 1, and abnormallyexpressed intron sequences such as N-acetylglucosaminyltransferase-V;clonal rearrangements of immunoglobulin genes generating uniqueidiotypes in myeloma and B-cell lymphomas; tumor antigens that includeepitopic regions or epitopic peptides derived from oncoviral processes,such as human papilloma virus proteins E6 and E7; Epstein bar virusprotein LMP2; nonmutated oncofetal proteins with a tumor-selectiveexpression, such as carcinoembryonic antigen and alpha-fetoprotein.

In other embodiments, an antigen is obtained or derived from apathogenic microorganism or from an opportunistic pathogenicmicroorganism (also called herein an infectious disease microorganism),such as a virus, fungus, parasite, and bacterium. In certainembodiments, antigens derived from such a microorganism includefull-length proteins.

Illustrative pathogenic organisms whose antigens are contemplated foruse in the method described herein include human immunodeficiency virus(HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV),cytomegalovirus (CMV), Epstein-Barr virus (EBV), Influenza A, B, and C,vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV),polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcusspecies including Methicillin-resistant Staphylococcus aureus (MRSA),and Streptococcus species including Streptococcus pneumoniae. As wouldbe understood by the skilled person, proteins derived from these andother pathogenic microorganisms for use as antigen as described hereinand nucleotide sequences encoding the proteins may be identified inpublications and in public databases such as GENBANK®, SWISS-PROT®, andTREMBL®.

Antigens derived from human immunodeficiency virus (HIV) include any ofthe HIV virion structural proteins (e.g., gp120, gp41, p17, p24),protease, reverse transcriptase, or HIV proteins encoded by tat, rev,nef, vif, vpr and vpu.

Antigens derived from herpes simplex virus (e.g., HSV 1 and HSV2)include, but are not limited to, proteins expressed from HSV late genes.The late group of genes predominantly encodes proteins that form thevirion particle. Such proteins include the five proteins from (UL) whichform the viral capsid: UL6, UL18, UL35, UL38 and the major capsidprotein UL19, UL45, and UL27, each of which may be used as an antigen asdescribed herein. Other illustrative HSV proteins contemplated for useas antigens herein include the ICP27 (H1, H2), glycoprotein B (gB) andglycoprotein D (gD) proteins. The HSV genome comprises at least 74genes, each encoding a protein that could potentially be used as anantigen.

Antigens derived from cytomegalovirus (CMV) include CMV structuralproteins, viral antigens expressed during the immediate early and earlyphases of virus replication, glycoproteins I and III, capsid protein,coat protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and1E2 (UL123 and UL122), protein products from the cluster of genes fromUL128-UL150 (Rykman, et al., 2006), envelope glycoprotein B (gB), gH,gN, and pp150. As would be understood by the skilled person, CMVproteins for use as antigens described herein may be identified inpublic databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see e.g.,Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

Antigens derived from Epstein-Ban virus (EBV) that are contemplated foruse in certain embodiments include EBV lytic proteins gp350 and gp110,EBV proteins produced during latent cycle infection includingEpstein-Ban nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C,EBNA-leader protein (EBNA-LP) and latent membrane proteins (LMP)-1,LMP-2A and LMP-2B (see, e.g., Lockey et al., 2008).

Antigens derived from respiratory syncytial virus (RSV) that arecontemplated for use herein include any of the eleven proteins encodedby the RSV genome, or antigenic fragments thereof: NS 1, NS2, N(nucleocapsid protein), M (Matrix protein) SH, G and F (viral coatproteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2(transcription regulation), RNA polymerase, and phosphoprotein P.

Antigens derived from Vesicular stomatitis virus (VSV) that arecontemplated for use include any one of the five major proteins encodedby the VSV genome, and antigenic fragments thereof: large protein (L),glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrixprotein (M) (see, e.g., Rieder et al., 1999).

Antigens derived from an influenza virus that are contemplated for usein certain embodiments include hemagglutinin (HA), neuraminidase (NA),nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1,PB1-F2, and PB2.

Exemplary viral antigens also include, but are not limited to,adenovirus polypeptides, alphavirus polypeptides, caliciviruspolypeptides (e.g., a calicivirus capsid antigen), coronaviruspolypeptides, distemper virus polypeptides, Ebola virus polypeptides,enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE)polypeptides (a hepatitis B core or surface antigen, a hepatitis C virusE1 or E2 glycoproteins, core, or non-structural proteins), herpesviruspolypeptides (including a herpes simplex virus or varicella zoster virusglycoprotein), infectious peritonitis virus polypeptides, leukemia viruspolypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides,papilloma virus polypeptides, parainfluenza virus polypeptides (e.g.,the hemagglutinin and neuraminidase polypeptides), paramyxoviruspolypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., a poliovirus capsid polypeptide), pox viruspolypeptides (e.g., a vaccinia virus polypeptide), rabies viruspolypeptides (e.g., a rabies virus glycoprotein G), reoviruspolypeptides, retrovirus polypeptides, and rotavirus polypeptides.

In certain embodiments, the antigen may be bacterial antigens. Incertain embodiments, a bacterial antigen of interest may be a secretedpolypeptide. In other certain embodiments, bacterial antigens includeantigens that have a portion or portions of the polypeptide exposed onthe outer cell surface of the bacteria.

Antigens derived from Staphylococcus species includingMethicillin-resistant Staphylococcus aureus (MRSA) that are contemplatedfor use include virulence regulators, such as the Agr system, Sar andSae, the Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU,SarV, SarX, SarZ and TcaR), the Srr system and TRAP. OtherStaphylococcus proteins that may serve as antigens include Clp proteins,HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g.,Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed.Jodi Lindsay). The genomes for two species of Staphylococcus aureus(N315 and Mu50) have been sequenced and are publicly available, forexample at PATRIC (PATRIC: The VBI PathoSystems Resource IntegrationCenter, Snyder et al., 2007). As would be understood by the skilledperson, Staphylococcus proteins for use as antigens may also beidentified in other public databases such as GenBank®, Swiss-Prot®, andTrEMBL®.

Antigens derived from Streptococcus pneumoniae that are contemplated foruse in certain embodiments described herein include pneumolysin, PspA,choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht,and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins ofStreptococcus pneumoniae are also known in the art and may be used as anantigen in some embodiments (see, e.g., Zysk et al., 2000). The completegenome sequence of a virulent strain of Streptococcus pneumoniae hasbeen sequenced and, as would be understood by the skilled person, S.pneumoniae proteins for use herein may also be identified in otherpublic databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins ofparticular interest for antigens according to the present disclosureinclude virulence factors and proteins predicted to be exposed at thesurface of the pneumococci (see, e.g., Frolet et al., 2010).

Examples of bacterial antigens that may be used as antigens include, butare not limited to, Actinomyces polypeptides, Bacillus polypeptides,Bacteroides polypeptides, Bordetella polypeptides, Bartonellapolypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA),Brucella polypeptides, Campylobacter polypeptides, Capnocytophagapolypeptides, Chlamydia polypeptides, Corynebacterium polypeptides,Coxiella polypeptides, Dermatophilus polypeptides, Enterococcuspolypeptides, Ehrlichia polypeptides, Escherichia polypeptides,Francisella polypeptides, Fusobacterium polypeptides, Haemobartonellapolypeptides, Haemophilus polypeptides (e.g., H. influenzae type b outermembrane protein), Helicobacter polypeptides, Klebsiella polypeptides,L-form bacteria polypeptides, Leptospira polypeptides, Listeriapolypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides,Neisseria polypeptides, Neorickettsia polypeptides, Nocardiapolypeptides, Pasteurella polypeptides, Peptococcus polypeptides,Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S.pneumoniae polypeptides) (see description herein), Proteus polypeptides,Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaeapolypeptides, Salmonella polypeptides, Shigella polypeptides,Staphylococcus polypeptides, group A streptococcus polypeptides (e.g.,S. pyogenes M proteins), group B streptococcus (S. agalactiae)polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Ypestis F1 and V antigens).

Examples of fungal antigens include, but are not limited to, Absidiapolypeptides, Acremonium polypeptides, Alternaria polypeptides,Aspergillus polypeptides, Basidiobolus polypeptides, Bipolarispolypeptides, Blastomyces polypeptides, Candida polypeptides,Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcuspolypeptides, Curvalaria polypeptides, Epidermophyton polypeptides,Exophiala polypeptides, Geotrichum polypeptides, Histoplasmapolypeptides, Madurella polypeptides, Malassezia polypeptides,Microsporum polypeptides, Moniliella polypeptides, Mortierellapolypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicilliumpolypeptides, Phialemonium polypeptides, Phialophora polypeptides,Prototheca polypeptides, Pseudallescheria polypeptides,Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidiumpolypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides,Sporothrix polypeptides, Stemphylium polypeptides, Trichophytonpolypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

Examples of protozoan parasite antigens include, but are not limited to,Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides,Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoonpolypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondiapolypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmaniapolypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosemapolypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides.Examples of helminth parasite antigens include, but are not limited to,Acanthocheilonema polypeptides, Aelurostrongylus polypeptides,Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascarispolypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillariapolypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosomapolypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides,Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydiumpolypeptides, Dirofilaria polypeptides, Dracunculus polypeptides,Enterobius polypeptides, Filaroides polypeptides, Haemonchuspolypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonellapolypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necatorpolypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides,Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagiapolypeptides, Parafilaria polypeptides, Paragonimus polypeptides,Parascaris polypeptides, Physaloptera polypeptides, Protostrongyluspolypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometrapolypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides,Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides,Toxocara polypeptides, Trichinella polypeptides, Trichostrongyluspolypeptides, Trichuris polypeptides, Uncinaria polypeptides, andWuchereria polypeptides. (e.g., P. falciparum circumsporozoite (PfCSP)),sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver stateantigen 1 (PfLSA1 c-term), and exported protein 1 (PfExp-1),Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosomapolypeptides, Theileria polypeptides, Toxoplasma polypeptides, andTrypanosoma polypeptides.

Examples of ectoparasite antigens include, but are not limited to,polypeptides (including antigens as well as allergens) from fleas;ticks, including hard ticks and soft ticks; flies, such as midges,mosquitoes, sand flies, black flies, horse flies, horn flies, deerflies, tsetse flies, stable flies, myiasis-causing flies and bitinggnats; ants; spiders, lice; mites; and true bugs, such as bed bugs andkissing bugs.

E. Suicide Genes

In some cases, any cells of the disclosure are modified to produce oneor more agents other than heterologous cytokines, engineered receptors,and so forth. In specific embodiments, the cells, such as NK cells, areengineered to harbor one or more suicide genes, and the term “suicidegene” as used herein is defined as a gene which, upon administration ofa prodrug, effects transition of a gene product to a compound whichkills its host cell. In some cases, the NK cell therapy may be subjectto utilization of one or more suicide genes of any kind when anindividual receiving the NK cell therapy and/or having received the NKcell therapy shows one or more symptoms of one or more adverse events,such as cytokine release syndrome, neurotoxicity, anaphylaxis/allergy,and/or on-target/off tumor toxicities (as examples) or is considered atrisk for having the one or more symptoms, including imminently. The useof the suicide gene may be part of a planned protocol for a therapy ormay be used only upon a recognized need for its use. In some cases thecell therapy is terminated by use of agent(s) that targets the suicidegene or a gene product therefrom because the therapy is no longerrequired.

Examples of suicide genes include engineered nonsecretable (includingmembrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides(see PCT/US19/62009, which is incorporated by reference herein in itsentirety), and they may be targeted by delivery of an antibody thatbinds the TNF-alpha mutant. Examples of suicide gene/prodrugcombinations that may be used are Herpes Simplex Virus-thymidine kinase(HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase andcycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinasethymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase andcytosine arabinoside. The E. coli purine nucleoside phosphorylase, aso-called suicide gene that converts the prodrug 6-methylpurinedeoxyriboside to toxic purine 6-methylpurine, may be utilized. Othersuicide genes include CD20, CD52, inducible caspase 9, purine nucleosidephosphorylase (PNP), Cytochrome p450 enzymes (CYP), Carboxypeptidases(CP), Carboxylesterase (CE), Nitroreductase (NTR), GuanineRibosyltransferase (XGRTP), Glycosidase enzymes, Methionine-α,γ-lyase(MET), and Thymidine phosphorylase (TP), as examples.

F. Methods of Delivery

One of skill in the art would be well-equipped to construct a vectorthrough standard recombinant techniques (see, for example, Sambrook etal., 2001 and Ausubel et al., 1996, both incorporated herein byreference) for the expression of the antigen receptors of the presentdisclosure. Vectors include but are not limited to, plasmids, cosmids,viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs), such as retroviral vectors (e.g.derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV,MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV,BIV, FIV etc.), adenoviral (Ad) vectors including replication competent,replication deficient and gutless forms thereof, adeno-associated viral(AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virusvectors, Epstein-Barr virus vectors, herpes virus vectors, vacciniavirus vectors, Harvey murine sarcoma virus vectors, murine mammary tumorvirus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectorsand group B adenovirus enadenotucirev vectors.

In specific embodiments, the vector is a multicistronic vector, such asis described in PCT/US19/62014, which is incorporated by referenceherein in its entirety. In such cases, a single vector may encode theCAR or TCR (and the expression construct may be configured in a modularformat to allow for interchanging parts of the CAR or TCR), a suicidegene, and one or more cytokines.

a. Viral Vectors

Viral vectors encoding an antigen receptor may be provided in certainaspects of the present disclosure. In generating recombinant viralvectors, non-essential genes are typically replaced with a gene orcoding sequence for a heterologous (or non-native) protein. A viralvector is a kind of expression construct that utilizes viral sequencesto introduce nucleic acid and possibly proteins into a cell. The abilityof certain viruses to infect cells or enter cells via receptormediated-endocytosis, and to integrate into host cell genomes andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Non-limiting examples of virus vectors that may beused to deliver a nucleic acid of certain aspects of the presentinvention are described below.

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, U.S. Pat. Nos. 6,013,516 and 5,994,136).

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell—wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat—is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference.

b. Regulatory Elements

Expression cassettes included in vectors useful in the presentdisclosure in particular contain (in a 5′-to-3′ direction) a eukaryotictranscriptional promoter operably linked to a protein-coding sequence,splice signals including intervening sequences, and a transcriptionaltermination/polyadenylation sequence. The promoters and enhancers thatcontrol the transcription of protein encoding genes in eukaryotic cellsare composed of multiple genetic elements. The cellular machinery isable to gather and integrate the regulatory information conveyed by eachelement, allowing different genes to evolve distinct, often complexpatterns of transcriptional regulation. A promoter used in the contextof the present disclosure includes constitutive, inducible, andtissue-specific promoters.

(i) Promoter/Enhancers

The expression constructs provided herein comprise a promoter to driveexpression of the antigen receptor. A promoter generally comprises asequence that functions to position the start site for RNA synthesis.The best known example of this is the TATA box, but in some promoterslacking a TATA box, such as, for example, the promoter for the mammalianterminal deoxynucleotidyl transferase gene and the promoter for the SV40late genes, a discrete element overlying the start site itself helps tofix the place of initiation. Additional promoter elements regulate thefrequency of transcriptional initiation. Typically, these are located inthe region 30110 bp-upstream of the start site, although a number ofpromoters have been shown to contain functional elements downstream ofthe start site as well. To bring a coding sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame “downstream” of (i.e., 3′ of)the chosen promoter. The “upstream” promoter stimulates transcription ofthe DNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the(lactamase (penicillinase), lactose and tryptophan (trp-) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein. Furthermore, it iscontemplated that the control sequences that direct transcription and/orexpression of sequences within non-nuclear organelles such asmitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally, any promoter/enhancer combination (as per, for example,the Eukaryotic Promoter Data Base EPDB, through world wide web atepd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7or SP6 cytoplasmic expression system is another possible embodiment.Eukaryotic cells can support cytoplasmic transcription from certainbacterial promoters if the appropriate bacterial polymerase is provided,either as part of the delivery complex or as an additional geneticexpression construct.

Non-limiting examples of promoters include early or late viralpromoters, such as, SV40 early or late promoters, cytomegalovirus (CMV)immediate early promoters, Rous Sarcoma Virus (RSV) early promoters;eukaryotic cell promoters, such as, e. g., beta actin promoter, GADPHpromoter, metallothionein promoter; and concatenated response elementpromoters, such as cyclic AMP response element promoters (cre), serumresponse element promoter (sre), phorbol ester promoter (TPA) andresponse element promoters (tre) near a minimal TATA box. It is alsopossible to use human growth hormone promoter sequences (e.g., the humangrowth hormone minimal promoter described at GenBank® Accession No.X05244, nucleotide 283-341) or a mouse mammary tumor promoter (availablefrom the ATCC, Cat. No. ATCC 45007). In certain embodiments, thepromoter is CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV,SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, howeverany other promoter that is useful to drive expression of the therapeuticgene is applicable to the practice of the present disclosure.

In certain aspects, methods of the disclosure also concern enhancersequences, i.e., nucleic acid sequences that increase a promoter'sactivity and that have the potential to act in cis, and regardless oftheir orientation, even over relatively long distances (up to severalkilobases away from the target promoter). However, enhancer function isnot necessarily restricted to such long distances as they may alsofunction in close proximity to a given promoter.

(ii) Initiation Signals and Linked Expression

A specific initiation signal also may be used in the expressionconstructs provided in the present disclosure for efficient translationof coding sequences. These signals include the ATG initiation codon oradjacent sequences. Exogenous translational control signals, includingthe ATG initiation codon, may need to be provided. One of ordinary skillin the art would readily be capable of determining this and providingthe necessary signals. It is well known that the initiation codon mustbe “in-frame” with the reading frame of the desired coding sequence toensure translation of the entire insert. The exogenous translationalcontrol signals and initiation codons can be either natural orsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements.

In certain embodiments, the use of internal ribosome entry sites (IRES)elements are used to create multigene, or polycistronic, messages. IRESelements are able to bypass the ribosome scanning model of 5′ methylatedCap dependent translation and begin translation at internal sites. IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described, as well an IRES from amammalian message. IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame is accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage.

Additionally, certain 2A sequence elements could be used to createlinked- or co-expression of genes in the constructs provided in thepresent disclosure. For example, cleavage sequences could be used toco-express genes by linking open reading frames to form a singlecistron. An exemplary cleavage sequence is the F2A (Foot-and-mouthdisease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A;T2A).

(iii) Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), for example, anucleic acid sequence corresponding to oriP of EBV as described above ora genetically engineered oriP with a similar or elevated function inprogramming, which is a specific nucleic acid sequence at whichreplication is initiated. Alternatively a replication origin of otherextra-chromosomally replicating virus as described above or anautonomously replicating sequence (ARS) can be employed.

c. Selection and Screenable Markers

In some embodiments, cells containing a construct of the presentdisclosure may be identified in vitro or in vivo by including a markerin the expression vector. Such markers would confer an identifiablechange to the cell permitting easy identification of cells containingthe expression vector. Generally, a selection marker is one that confersa property that allows for selection. A positive selection marker is onein which the presence of the marker allows for its selection, while anegative selection marker is one in which its presence prevents itsselection. An example of a positive selection marker is a drugresistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selection markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes as negative selection markers such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beutilized. One of skill in the art would also know how to employimmunologic markers, possibly in conjunction with FACS analysis. Themarker used is not believed to be important, so long as it is capable ofbeing expressed simultaneously with the nucleic acid encoding a geneproduct. Further examples of selection and screenable markers are wellknown to one of skill in the art.

d. Other Methods of Nucleic Acid Delivery

In addition to viral delivery of the nucleic acids encoding the antigenreceptor, the following are additional methods of recombinant genedelivery to a given host cell and are thus considered in the presentdisclosure.

Introduction of a nucleic acid, such as DNA or RNA, into the immunecells of the current disclosure may use any suitable methods for nucleicacid delivery for transformation of a cell, as described herein or aswould be known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, direct delivery of DNA such as by exvivo transfection, by injection, including microinjection); byelectroporation; by calcium phosphate precipitation; by usingDEAE-dextran followed by polyethylene glycol; by direct sonic loading;by liposome mediated transfection and receptor-mediated transfection; bymicroprojectile bombardment; by agitation with silicon carbide fibers;by Agrobacterium-mediated transformation; bydesiccation/inhibition-mediated DNA uptake, and any combination of suchmethods. Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

VI. METHODS OF TREATMENT

Embodiments of the disclosure include methods of treating an individualfor cancer, infections of any kind, and any immune disorder. Theindividual may utilize the treatment method of the disclosure as aninitial treatment or after another treatment or during anothertreatment. The immunotherapy methods may be tailored to the need of anindividual with cancer based on the type or stage of cancer, and in atleast some cases the immunotherapy may be modified during the course oftreatment for the individual.

In specific cases, the treatment methods are as follows: 1) Adoptivecellular therapy with T or NK cells (ex vivo expanded or expressing CARsor TCRs) to treat cancer patients with any type of hematologicmalignancy, (2) Adoptive cellular therapy with T or NK cells (ex vivoexpanded or expressing CARs or TCRs) to treat cancer patients with anytype of solid cancers, (3) Adoptive cellular therapy (ex vivo expandedor expressing CARs or TCRs) with Tregs and regulatory B cells to treatpatients with immune disorders, (4) Adoptive cellular therapy with T orNK cells (ex vivo expanded or expressing CARs or TCRs) to treat patientswith infectious diseases. The present disclosure is the first to showthat knocking down/out multiple genes in human NK cells contributes tothe cell's improved functionality and resistance to tumormicroenvironment. In specific embodiments, this has direct implicationson patient care using a novel immunotherapeutic approach that enhancesthe function of a patient's own immune cells or adoptively transferredimmune cells. Embodiments provide a novel approach to produce highlyfunctional T, NK and B cells (both ex vivo expanded and CAR or TCRengineered) for immunotherapy. These include targeting cancers—bothhematologic and solid tumors-(NK cells and T cells, also CAR T cells andCAR NK cells), autoimmune and alloimmune disorders (B cells, regulatoryB cells and regulatory T cells) and treatment of infections (forpathogen-specific T cells).

In some embodiments, the present disclosure provides methods forimmunotherapy comprising administering an effective amount of the immunecells of the present disclosure. In one embodiment, a medical disease ordisorder is treated by transfer of immune NK cell population thatelicits an immune response. In certain embodiments of the presentdisclosure, cancer or infection is treated by transfer of an immune cellpopulation that elicits an immune response. Provided herein are methodsfor treating or delaying progression of cancer in an individualcomprising administering to the individual an effective amount anantigen-specific cell therapy. The present methods may be applied forthe treatment of immune disorders, solid cancers, hematologic cancers,and viral infections.

Tumors for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to, a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, lung cancer (includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung), cancer of the peritoneum,gastric or stomach cancer (including gastrointestinal cancer andgastrointestinal stromal cancer), pancreatic cancer, cervical cancer,ovarian cancer, liver cancer, bladder cancer, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, various types of head and neck cancer, and melanoma.

The cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; lentigomalignant melanoma; acral lentiginous melanomas; nodular melanomas;malignant melanoma in giant pigmented nevus; epithelioid cell melanoma;blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; B-cell lymphoma; low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignanthistiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferativesmall intestinal disease; leukemia; lymphoid leukemia; plasma cellleukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloidleukemia; basophilic leukemia; eosinophilic leukemia; monocyticleukemia; mast cell leukemia; megakaryoblastic leukemia; myeloidsarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronicmyeloblastic leukemia.

Particular embodiments concern methods of treatment of leukemia.Leukemia is a cancer of the blood or bone marrow and is characterized byan abnormal proliferation (production by multiplication) of blood cells,usually white blood cells (leukocytes). It is part of the broad group ofdiseases called hematological neoplasms. Leukemia is a broad termcovering a spectrum of diseases. Leukemia is clinically andpathologically split into its acute and chronic forms.

In certain embodiments of the present disclosure, immune cells aredelivered to an individual in need thereof, such as an individual thathas cancer or an infection. The cells then enhance the individual'simmune system to attack the respective cancer or pathogenic cells. Insome cases, the individual is provided with one or more doses of theimmune cells. In cases where the individual is provided with two or moredoses of the immune cells, the duration between the administrationsshould be sufficient to allow time for propagation in the individual,and in specific embodiments the duration between doses is 1, 2, 3, 4, 5,6, 7, or more days.

Certain embodiments of the present disclosure provide methods fortreating or preventing an immune-mediated disorder. In one embodiment,the subject has an autoimmune disease. Non-limiting examples ofautoimmune diseases include: alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunediseases of the adrenal gland, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgAneuropathy, juvenile arthritis, lichen planus, lupus erthematosus,Meniere's disease, mixed connective tissue disease, multiple sclerosis,type 1 or immune-mediated diabetes mellitus, myasthenia gravis,nephrotic syndrome (such as minimal change disease, focalglomerulosclerosis, or mebranous nephropathy), pemphigus vulgaris,pernicious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, ulcerativecolitis, uveitis, vasculitides (such as polyarteritis nodosa, takayasuarteritis, temporal arteritis/giant cell arteritis, or dermatitisherpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus,some examples of an autoimmune disease that can be treated using themethods disclosed herein include, but are not limited to, multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosis, type Idiabetes mellitus, Crohn's disease; ulcerative colitis, myastheniagravis, glomerulonephritis, ankylosing spondylitis, vasculitis, orpsoriasis. The subject can also have an allergic disorder such asAsthma.

In yet another embodiment, the subject is the recipient of atransplanted organ or stem cells and immune cells are used to preventand/or treat rejection. In particular embodiments, the subject has or isat risk of developing graft versus host disease. GVHD is a possiblecomplication of any transplant that uses or contains stem cells fromeither a related or an unrelated donor. There are two kinds of GVHD,acute and chronic. Acute GVHD appears within the first three monthsfollowing transplantation. Signs of acute GVHD include a reddish skinrash on the hands and feet that may spread and become more severe, withpeeling or blistering skin. Acute GVHD can also affect the stomach andintestines, in which case cramping, nausea, and diarrhea are present.Yellowing of the skin and eyes (jaundice) indicates that acute GVHD hasaffected the liver. Chronic GVHD is ranked based on its severity:stage/grade 1 is mild; stage/grade 4 is severe. Chronic GVHD developsthree months or later following transplantation. The symptoms of chronicGVHD are similar to those of acute GVHD, but in addition, chronic GVHDmay also affect the mucous glands in the eyes, salivary glands in themouth, and glands that lubricate the stomach lining and intestines. Anyof the populations of immune cells disclosed herein can be utilized.Examples of a transplanted organ include a solid organ transplant, suchas kidney, liver, skin, pancreas, lung and/or heart, or a cellulartransplant such as islets, hepatocytes, myoblasts, bone marrow, orhematopoietic or other stem cells. The transplant can be a compositetransplant, such as tissues of the face. Immune cells can beadministered prior to transplantation, concurrently withtransplantation, or following transplantation. In some embodiments, theimmune cells are administered prior to the transplant, such as at least1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least1 month prior to the transplant. In one specific, non-limiting example,administration of the therapeutically effective amount of immune cellsoccurs 3-5 days prior to transplantation.

In some embodiments, the subject can be administered nonmyeloablativelymphodepleting chemotherapy prior to the immune cell therapy. Thenonmyeloablative lymphodepleting chemotherapy can be any suitable suchtherapy, which can be administered by any suitable route. Thenonmyeloablative lymphodepleting chemotherapy can comprise, for example,the administration of cyclophosphamide and fludarabine, particularly ifthe cancer is melanoma, which can be metastatic. An exemplary route ofadministering cyclophosphamide and fludarabine is intravenously.Likewise, any suitable dose of cyclophosphamide and fludarabine can beadministered. In particular aspects, around 60 mg/kg of cyclophosphamideis administered for two days after which around 25 mg/m² fludarabine isadministered for five days.

In certain embodiments, a growth factor that promotes the growth andactivation of the immune cells is administered to the subject eitherconcomitantly with the immune cells or subsequently to the immune cells.The immune cell growth factor can be any suitable growth factor thatpromotes the growth and activation of the immune cells. Examples ofsuitable immune cell growth factors include interleukin (IL)-2, IL-7,IL-12, IL-15, IL-18, and IL-21, which can be used alone or in variouscombinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15,IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

Therapeutically effective amounts of immune cells can be administered bya number of routes, including parenteral administration, for example,intravenous, intraperitoneal, intramuscular, intrasternal, orintraarticular injection, or infusion.

The therapeutically effective amount of immune cells for use in adoptivecell therapy is that amount that achieves a desired effect in a subjectbeing treated. For instance, this can be the amount of immune cellsnecessary to inhibit advancement, or to cause regression of anautoimmune or alloimmune disease, or which is capable of relievingsymptoms caused by an autoimmune disease, such as pain and inflammation.It can be the amount necessary to relieve symptoms associated withinflammation, such as pain, edema and elevated temperature. It can alsobe the amount necessary to diminish or prevent rejection of atransplanted organ.

The immune cell population can be administered in treatment regimensconsistent with the disease, for example a single or a few doses overone to several days to ameliorate a disease state or periodic doses overan extended time to inhibit disease progression and prevent diseaserecurrence. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. The therapeuticallyeffective amount of immune cells will be dependent on the subject beingtreated, the severity and type of the affliction, and the manner ofadministration. In some embodiments, doses that could be used in thetreatment of human subjects range from at least 3.8×10⁴, at least3.8×10⁵, at least 3.8×10⁶, at least 3.8×10⁷, at least 3.8×10⁸, at least3.8×10⁹, or at least 3.8×10¹⁰ immune cells/m². In a certain embodiment,the dose used in the treatment of human subjects ranges from about3.8×10⁹ to about 3.8×10¹⁰ immune cells/m². In additional embodiments, atherapeutically effective amount of immune cells can vary from about5×10⁶ cells per kg body weight to about 7.5×10⁸ cells per kg bodyweight, such as about 2×10⁷ cells to about 5×10⁸ cells per kg bodyweight, or about 5×10⁷ cells to about 2×10⁸ cells per kg body weight.The exact amount of immune cells is readily determined by one of skillin the art based on the age, weight, sex, and physiological condition ofthe subject. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The immune cells may be administered in combination with one or moreother therapeutic agents for the treatment of the immune-mediateddisorder. Combination therapies can include, but are not limited to, oneor more anti-microbial agents (for example, antibiotics, anti-viralagents and anti-fungal agents), anti-tumor agents (for example,fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide,doxorubicin, or vincristine), immune-depleting agents (for example,fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressiveagents (for example, azathioprine, or glucocorticoids, such asdexamethasone or prednisone), anti-inflammatory agents (for example,glucocorticoids such as hydrocortisone, dexamethasone or prednisone, ornon-steroidal anti-inflammatory agents such as acetylsalicylic acid,ibuprofen or naproxen sodium), cytokines (for example, interleukin-10 ortransforming growth factor-beta), hormones (for example, estrogen), or avaccine. In addition, immunosuppressive or tolerogenic agents includingbut not limited to calcineurin inhibitors (e.g., cyclosporin andtacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil,antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or Bcells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan,Busulfan); irradiation; or chemokines, interleukins or their inhibitors(e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can beadministered. Such additional pharmaceutical agents can be administeredbefore, during, or after administration of the immune cells, dependingon the desired effect. This administration of the cells and the agentcan be by the same route or by different routes, and either at the samesite or at a different site.

A. Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions and formulationscomprising immune cells (e.g., T cells, B cells, or NK cells) and apharmaceutically acceptable carrier.

Pharmaceutical compositions and formulations as described herein can beprepared by mixing the active ingredients (such as an antibody or apolypeptide) having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 22^(nd) edition, 2012), in the form oflyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn— protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

B. Combination Therapies

In certain embodiments, the compositions and methods of the presentembodiments involve an immune cell population in combination with atleast one additional therapy. The additional therapy may be radiationtherapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, genetherapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bonemarrow transplantation, nanotherapy, monoclonal antibody therapy, or acombination of the foregoing. The additional therapy may be in the formof adjuvant or neoadjuvant therapy.

In some embodiments, the additional therapy is the administration ofsmall molecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy istherapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulininhibitor, apoptosis inhibitor, and/or chemopreventative agent. Theadditional therapy may be one or more of the chemotherapeutic agentsknown in the art.

An immune cell therapy may be administered before, during, after, or invarious combinations relative to an additional cancer therapy, such asimmune checkpoint therapy. The administrations may be in intervalsranging from concurrently to minutes to days to weeks. In embodimentswhere the immune cell therapy is provided to a patient separately froman additional therapeutic agent, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the two compounds would still be able to exert anadvantageously combined effect on the patient. In such instances, it iscontemplated that one may provide a patient with the antibody therapyand the anti-cancer therapy within about 12 to 24 or 72 h of each otherand, more particularly, within about 6-12 h of each other. In somesituations it may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

Various combinations may be employed. For the example below an immunecell therapy is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

1. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PS Kpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as y-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation, and UV-irradiation. It is most likely that all of thesefactors affect a broad range of damage on DNA, on the precursors of DNA,on the replication and repair of DNA, and on the assembly andmaintenance of chromosomes. Dosage ranges for X-rays range from dailydoses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk),to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

3. Immunotherapy

The skilled artisan will understand that additional immunotherapies maybe used in combination or in conjunction with methods of theembodiments. In the context of cancer treatment, immunotherapeutics,generally, rely on the use of immune effector cells and molecules totarget and destroy cancer cells. Rituximab (RITUXAN®) is such anexample. The immune effector may be, for example, an antibody specificfor some marker on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually affect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells

Antibody—drug conjugates (ADCs) comprise monoclonal antibodies (MAbs)that are covalently linked to cell-killing drugs and may be used incombination therapies. This approach combines the high specificity ofMAbs against their antigen targets with highly potent cytotoxic drugs,resulting in “armed” MAbs that deliver the payload (drug) to tumor cellswith enriched levels of the antigen. Targeted delivery of the drug alsominimizes its exposure in normal tissues, resulting in decreasedtoxicity and improved therapeutic index. Exemplary ADC drugs includeADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine orT-DM1).

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies include immune adjuvants, e.g.,Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, andaromatic compounds); cytokine therapy, e.g., interferons α, β, and γ,IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; andmonoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, andanti-p185. It is contemplated that one or more anti-cancer therapies maybe employed with the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpointinhibitor. Immune checkpoints either turn up a signal (e.g.,co-stimulatory molecules) or turn down a signal. Inhibitory immunecheckpoints that may be targeted by immune checkpoint blockade includeadenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and Tlymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO),killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3),programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). Inparticular, the immune checkpoint inhibitors target the PD-1 axis and/orCTLA-4.

The immune checkpoint inhibitors may be drugs such as small molecules,recombinant forms of ligand or receptors, or, in particular, areantibodies, such as human antibodies. Known inhibitors of the immunecheckpoint proteins or analogs thereof may be used, in particularchimerized, humanized or human forms of antibodies may be used. As theskilled person will know, alternative and/or equivalent names may be inuse for certain antibodies mentioned in the present disclosure. Suchalternative and/or equivalent names are interchangeable in the contextof the present disclosure. For example it is known that lambrolizumab isalso known under the alternative and equivalent names MK-3475 andpembrolizumab.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2.In another embodiment, a PDL1 binding antagonist is a molecule thatinhibits the binding of PDL1 to its binding partners. In a specificaspect, PDL1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PDL2 binding antagonist is a molecule that inhibits thebinding of PDL2 to its binding partners. In a specific aspect, a PDL2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody). In some embodiments, the anti-PD-1 antibody is selected fromthe group consisting of nivolumab, pembrolizumab, and CT-011. In someembodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPDL1 or PDL2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 bindingantagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106,ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may beused. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011,also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224,also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

Another immune checkpoint that can be targeted in the methods providedherein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), alsoknown as CD152. The complete cDNA sequence of human CTLA-4 has theGenbank accession number L15006. CTLA-4 is found on the surface of Tcells and acts as an “off” switch when bound to CD80 or CD86 on thesurface of antigen-presenting cells. CTLA4 is a member of theimmunoglobulin superfamily that is expressed on the surface of Helper Tcells and transmits an inhibitory signal to T cells. CTLA4 is similar tothe T-cell co-stimulatory protein, CD28, and both molecules bind to CD80and CD86, also called B7-1 and B7-2 respectively, on antigen-presentingcells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28transmits a stimulatory signal. Intracellular CTLA4 is also found inregulatory T cells and may be important to their function. T cellactivation through the T cell receptor and CD28 leads to increasedexpression of CTLA-4, an inhibitory receptor for B7 molecules.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody), an antigen binding fragment thereof, an immunoadhesin, afusion protein, or oligopeptide.

Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom)suitable for use in the present methods can be generated using methodswell known in the art. Alternatively, art recognized anti-CTLA-4antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab(also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen bindingfragments and variants thereof. In other embodiments, the antibodycomprises the heavy and light chain CDRs or VRs of ipilimumab.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 andCDR3 domains of the VL region of ipilimumab. In another embodiment, theantibody competes for binding with and/or binds to the same epitope onCTLA-4 as the above-mentioned antibodies. In another embodiment, theantibody has at least about 90% variable region amino acid sequenceidentity with the above-mentioned antibodies (e.g., at least about 90%,95%, or 99% variable region identity with ipilimumab).

4. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

5. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

VII. ARTICLES OF MANUFACTURE OR KITS

An article of manufacture or a kit is provided comprising immune cellsis also provided herein. The article of manufacture or kit can furthercomprise a package insert comprising instructions for using the immunecells to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of theantigen-specific immune cells described herein may be included in thearticle of manufacture or kits. Suitable containers include, forexample, bottles, vials, bags and syringes. The container may be formedfrom a variety of materials such as glass, plastic (such as polyvinylchloride or polyolefin), or metal alloy (such as stainless steel orhastelloy). In some embodiments, the container holds the formulation andthe label on, or associated with, the container may indicate directionsfor use. The article of manufacture or kit may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. In some embodiments, the article ofmanufacture further includes one or more of another agent (e.g., achemotherapeutic agent, and anti-neoplastic agent). Suitable containersfor the one or more agent include, for example, bottles, vials, bags andsyringes.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Multiplex Gene Editing

To test the efficacy of simultaneously disrupting the expression ofmultiple genes in immune cells, such as T cell and NK cells, severalstudies were performed to test the disruption of different combinationsof genes using CRISPR. In a first study, CRISPR/Cas9 was used to disruptexpression of NKG2A, CD47, TGFBR2, and CISH in NK cells. In this set ofgenes, NKG2A and CD47 were knocked out in the first round ofelectroporation and in the second round of electroporation CISH andTGFBR2 were targeted. Knockout efficiency was successfully validatedusing PCR and flow-cytometry for both rounds of electroporation (FIG.1).

The method of disrupting multiple genes was validated in additional setsof genes including TIGIT, CD96, CISH, Adenosine (FIG. 2) and NKG2A,CD47, TGFBR2 and CISH (FIG. 3). It was found that the disruption of themultiple genes results in enhanced functionality against target tumorcells. Flow cytometric analysis of IFN-γ, TNFα and CD107 production wasperformed with varying NK cells (Edited vs Cas9 alone) co-stimulatedwith target cell lines for 5 hr in the presence of Brefeldin A. Therewas enhanced IFN-γ, TNFα and CD107 secretion following stimulation withtarget cell lines (FIG. 3)

This enhanced functionality was confirmed by disruption of NKG2A, CD47,TGFBR2 and CISH in NK cells which showed enhanced antitumorcytotoxicity. (FIG. 4A) as measured by ⁵¹Cr-release assay, against K562cells. In addition, following 30 minutes of recombinant TGF-B treatment(50 ng/ml) pSMAD activity was measured by flow cytometry (FIG. 4B). Itwas also observed that NK cells lose CD16 and CD62L expression uponcytokine stimulation or target recognition (FIG. 5) and knockout ofADAM17 in NK cells prevent shedding of CD16 and CD62L (FIG. 6) andimproves ADCC and cytotoxicity against K562 targets (FIG. 7).

Further studies showed that disruption of SHP1 in NK cells leads toenhanced antitumor efficacy (FIGS. 9 and 10). NK cells were co-culturedwith K562\Raji cells at a 1:1 ratio for 4 hours. After the incubation,the cells were stained with annexin V and live and dead cells wereanalyzed. The K562 cells are sensitive to NK cell killing and the Rajicells are resistant to NK cell killing. In addition, disruption of NKG2Ain NK-CAR cells led to enhanced antitumor efficacy against Raji targets(FIG. 12).

The present approach was further validated with additional sets ofgenes—TIGIT, CD96, CISH, and ADENOSINE as well as NKG2A, CISH, TGFBRIIand ADENOSINE. NK cell function was evaluated by flow cytometricmeasurement and an increase was observed in TNFα, IFNγ, and CD107a inthe cells upon target cell line stimulation (FIGS. 13-14).

Thus, the present methods can be used to simultaneously disruptexpression of multiple genes in immune cells to increase theirfunctionality.

Example 2—Methods

sgRNA-Cas9 pre-complexing and Electroporation: 1 or 2 sgRNAs spanningclose regions were designed and used for each gene. 1 ug cas9 (PNA Bio)and 500 ng sgRNA (sum of all sgRNAs) reactions were made for each geneand incubated on ice for 20 minutes. After 20 minutes, 250,000 NK Cellswere re-suspended in T-buffer* (included with Neon Electroporation Kit,Invitrogen, total volume including RNP complex and cells should be 14ul) and electroporated with 10 ul electroporation tip using NeonTransfection System. The electroporation conditions are 1600V, 10 ms,and 3 pulses* for NK cells. The cells were then added to culture platewith APCs (1 NK: 2 APCs), SCGM media (preferentially antibiotic free),2001U/ml IL2 and allowed to recover in 37° C. incubator.

crRNA pre-complexing and Electroporation: The crRNA and tracrRNA duplexwas mixed with a pipette and centrifuges. The mixture was incubated at95° C. for 5 min in a thermocycler and then allowed to cool to roomtemperature on the benchtop.

TABLE 3 crNRA and tracrRNA duplex. volume concentration volumeconcentration crRNA # 1 2.2 ul 100 uM crRNA # 2 2.2 ul 100 uM tracrRNA2.2 ul 100 uM tracrRNA 2.2 ul 100 uM IDTE Buffer 5.6 ul IDTE Buffer 5.6ul total volume 10 ul 44 uM total volume 10 ul 44 uMThe starting concentration of crRNA and tracrRNA are 100 uM. The finalconcentration after mixing them in equimolar concentration is 44 uM.

TABLE 4 Cas9 Nuclease. volume Alt-R S.p. Cas9 Nuclease 3 ul 3NLS (61 uM)T buffer 7 ul total volume 10 ul final concentration 18 uM

TABLE 5 Combination of the crRNA: tracrRNA and Cas9 nuclease mix. volumecrRNA # 1: tracrRNA duplex (Step 1) 2 ul Cas9 (Step 2) 2 ul total volume4 ul

The crRNA: tracrRNA duplex was combined with the Cas9 Nuclease Mix withpipette, and incubated at room temperature for 15 min. The mixture wasthen combined with crRNA.

TABLE 6 crRNA # 1 and crRNA # 2. volume crRNA # 1 + tracrRNA + cas9(Step 3) 2.25 ul crRNA # 2 + tracrRNA + cas9 (Step 3) 2.25 ul totalvolume 4.5 ul

Electroporation was performed by first preparing culture plates withAPCs (1 NK: 2 APCs), SCGM media (preferably antibiotic free), and2001U/mL IL-2. Prepare 250,000 cells per well were re-suspended in 7.5ul T buffer just before use. The electroporation conditions were 1600V,10 ms, and 3 pulses*. The cells were then added to culture plate andallowed to recover in a 37° C. incubator.

NK Cell expansion: Isolate NK cells from cord blood or peripheral bloodusing NK cell isolation kit from Miltenyi (130-092-657). Put NK cellswith Feeder cell at 1:2 ratio (1 NK cell 2 Feeder cells) in the presenceof IL2 (200 IU\ml) in SCGM media. Change media every other day with IL2.On day 4 reselect NK cells using NK cell isolation KIT to remove feedercells or wait for day 7 until all feeder cells are gone. Transduce withchimeric antigen receptor or electroporate for Crispr-Cas9.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An in vitro method for the disruption of at leasttwo genes in an immune cell, wherein the at least two genes are selectedfrom the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1,TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA,SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86,IL10R, CD5, and CD7.
 2. The method of claim 1, wherein disruptioncomprises introducing a guide RNA (gRNA) for each gene to said immunecell.
 3. The method of claim 1 or 2, wherein the at least two genes areselected from the group consisting of (a) NKG2A and CISH, (b) NKG2A andTGFBRII, (c) CISH and TGFBRII, (d) TIGIT and FOXO1, (e) TIGIT andTGFBRII, (f) CD96 and FOXO1, (g) CD96 and TGFBRII, (h) FOXO1 andTGFBRII, (i) CD96 and TIGIT, (j) CISH and TIGIT, (k) TIM3 and CISH, (l)TIM3 and TGFBRII, (m) FOXO1 and TGFBRII, (n) TIM3 and TIGIT, (o) SIGLEC7and CISH, (p) SIGLEC7 and TGFBRII, (q) CD47 and CISH, (r) CD47 andTGFBRII, (s) SIRPA and CISH, (t) SIRPA and TGFBRII, (u) CD47 and TIGIT,(v) CD47 and SIRPA, (w) A2AR and CISH, (x) A2AR and TGFBRII, (y) ADAM17and CISH, (z) TGFBRII and ADAM17, (al) A2AR and TIGIT, (b1) SHP1 andCISH, (c1) CISH and TGFBRII, (d1) SHP1 and TGFBRII, (e1) SHP1 and TIGIT,and (f1) SHP1 and TIM3.
 4. The method of any of claims 1-3, wherein atleast 3 genes are disrupted.
 5. The method of claim 4, wherein the atleast 3 genes are selected from the group consisting of (1) NKG2A, CISH,and TGFBRII, (2) TIGIT, FOXO1, and TGFBRII, (3) TGFBRII, CD96, andTIGIT, (4) TGFBR2, CISH, and TIGIT, (5) TIM3, CISH, and TGFBRII, (6)CD96, FOXO1, and TGFBRII, (7) TGFBRII, TIM3, and TIGIT, (8) SIGLEC7,CISH, and TGFBRII, (9) CD47, CISH, and TGFBRII, (10) SIRPA, CISH, andTGFBRII, (11) TGFBRII, CD47, and TIGIT, (12) TGFBRII, CD47, and SIRPA,(13) A2AR, CISH, and TGFBRII, (14) TGFBRII, CISH, and ADAM17, (15)TGFBRII, TIM3, and TIGIT, (16) TGFBRII, A2AR, and TIGIT, (17) SHP1,CISH, and TGFBRII, (18) TGFBRII, CISH, and SHP1, (19) TGFBRII, SHP1, andTIGIT, and (20) TGFBRII, SHP1, and TIM3.
 6. The method of claim 2,further comprising introducing an RNA-guide endonuclease.
 7. The methodof claim 6, wherein the RNA-guided endonuclease is Cas9.
 8. The methodof claim 6, wherein introducing the RNA-guided endonuclease comprisesintroducing a nucleic acid encoding the RNA-guided endonuclease into theimmune cell.
 9. The method of claim 8, wherein the nucleic acid is mRNA.10. The method of any of claims 1-9, wherein three, four, five, or sixgenes are disrupted.
 11. The method of claim 10, wherein the genescomprise two of (a)-(j1) subgroups.
 12. The method of claim 10, whereinthe genes comprise one subgroup of (a)-(j1) and one subgroup of 1-23.13. The method of claim 10, wherein the genes comprise two of 1-23subgroups.
 14. The method of claim 1, wherein the disruption issimultaneous.
 15. The method of any of claims 1-14, wherein the immunecell is a T cell, NK cell, NK T cell, B cell, or stem cell.
 16. Themethod of claim 15, wherein the immune cell is engineered to express achimeric antigen receptor (CAR) and/or T cell receptor (TCR).
 17. Themethod of claim 15, wherein the immune cell is engineered to express aCAR.
 18. The method of claim 15, wherein the immune cell is engineeredto express a TCR.
 19. The method of claim 15, wherein the immune cell isengineered to express a CAR and TCR.
 20. The method of claim 15, whereinthe immune cell is virus-specific.
 21. The method of claim 15, whereinthe T cell is a virus-specific T cell.
 22. The method of claim 15,wherein the T cell is a regulatory T cell.
 23. The method of claim 15,wherein the B cell is a regulatory B cell.
 24. The method of claim 15,wherein the stem cell is a mesenchymal stem cell (MSC) or an inducedpluripotent stem (iPS) cell.
 25. The method of claim 15, wherein the Tcell is a CD8⁺ T cell, CD4⁺ T cell, gamma-delta T cell, or mixturethereof.
 26. The method of any of claims 1-25, wherein the immune cellis isolated from peripheral blood, cord blood, bone marrow or a mixturethereof.
 27. The method of any one of claims 1-26, wherein the immunecell is isolated from cord blood.
 28. The method of claim 27, whereinthe cord blood is pooled from 2 or more individual cord blood units. 29.The method of any of claims 2-28, wherein introducing comprisestransfecting or transducing.
 30. The method of any of claims 2-28,wherein introducing comprises electroporation.
 31. The method of claim30, wherein electroporation is performed more than once.
 32. The methodof claim 31, wherein two rounds of electroporation are performed. 33.The method of claim 32, wherein a first group of CRISPR gRNAs areintroduced in a first electroporation and a second group of CRISPR gRNAsare introduced in a second round of electroporation.
 34. The method ofclaim 33, wherein the first group and/or second group of CRISPR gRNAscomprise 1, 2, 3, or 4 CRISPR gRNAs.
 35. The method of claim 32, whereintwo CRISPR gRNAs are introduced in a first electroporation and twoCRISPR gRNAs are introduced in a second round of electroporation. 36.The method of any one of claims 1-35, wherein the method comprisesdisrupting NKG2A, CD47, TGFβR2, and CISH.
 37. The method of any one ofclaims 1-35, wherein the method comprises disrupting NKG2A, CISH, TGFβR2and ADORA2.
 38. The method of any one of claims 1-35, wherein the methodcomprises disrupting NKG2A, TGFβR2 and CISH.
 39. The method of any oneof claims 1-35, wherein the method comprises disrupting TIGIT, CD96,CISH, and ADORA2.
 40. The method of any one of claims 1-35, wherein themethod comprises disrupting ADAM17, TGFβR2 NKG2A and SHP1.
 41. Themethod of any of claims 1-40, wherein the disruption results in enhancedantitumor cytotoxicity, in vivo proliferation, in vivo persistence,and/or improved function of the immune cell.
 42. The method of claim 41,wherein the immune cell has increased secretion of IFN-γ, CD107, and/orTNFα.
 43. The method of claim 41, wherein the immune cell has increasedproduction of perforin and/or granzyme B.
 44. The method of any ofclaims 1-43, further comprising introducing a CAR or TCR to said immunecell.
 45. The method of claim 44, wherein introducing comprisesintroducing a nucleic acid encoding said CAR or TCR into said immunecell.
 46. The method of claim 45, wherein the nucleic acid is in anexpression vector.
 47. The method of claim 46, wherein the expressionvector is a retroviral vector.
 48. The method of claim 47, wherein theretroviral vector is an adenovirus-associated vector.
 49. The method ofclaim 48, wherein the adenovirus-associated vector is AAV6.
 50. Themethod of claim 46, wherein the vector further comprises an inhibitorygene sequence.
 51. The method of claim 50, wherein the inhibitory genesequence is selected from the group consisting of NKG2A, SIGLEC-7, LAG3,TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1,PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R,ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7.
 52. The method of claim50, wherein the vector further comprises a guide RNA for said inhibitorygene.
 53. The method of claim 52, wherein the CAR is flanked by homologyarms for said inhibitory gene.
 54. The method of claim 53, whereinintroducing the vector comprising the CAR sequence results in insertionof the CAR at the inhibitory gene locus in said immune cell.
 55. Themethod of claim 54, wherein the CAR is inserted at an exon of saidinhibitory gene.
 56. The method of claim 54, wherein the CAR is underthe control of the endogenous promoter of the inhibitory gene.
 57. Themethod of claim 54, wherein introducing the vector further disruptsexpression of said inhibitory gene.
 58. An immune cell with disruptedexpression of at least two genes in the immune cell, wherein the atleast two genes are selected from the group consisting of NKG2A,SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1,PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40,IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7.
 59. The cell ofclaim 58, wherein the cell is produced according to any one of claims1-57.
 60. The cell of claim 58, wherein three, four, five, or six genesare disrupted.
 61. The cell of claim 58, wherein the immune cell is a Tcell, NK cell, NK T cell, B cell, or stem cell.
 62. The cell of claim58, wherein the immune cell is engineered to express a chimeric antigenreceptor (CAR) and/or T cell receptor (TCR).
 63. The cell of claim 58,wherein the immune cell is engineered to express a CAR.
 64. The cell ofclaim 58, wherein the immune cell is engineered to express a TCR. 65.The cell of claim 58, wherein the immune cell is engineered to express aCAR and TCR.
 66. The cell of claim 58, wherein the immune cell isvirus-specific.
 67. The cell of claim 61, wherein the T cell is avirus-specific T cell.
 68. The cell of claim 61, wherein the T cell is aregulatory T cell.
 69. The cell of claim 61, wherein the B cell is aregulatory B cell.
 70. The cell of claim 61, wherein the stem cell is amesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell.71. The cell of claim 61, wherein the T cell is a CD8⁺ T cell, CD4⁺ Tcell, or gamma-delta T cell.
 72. The cell of claim 58, wherein theimmune cell is isolated from peripheral blood, cord blood, bone marrow,or a mixture thereof.
 73. The cell of claim 58, wherein the immune cellis isolated from cord blood.
 74. The cell of claim 73, wherein the cordblood is pooled from 2 or more individual cord blood units.
 75. The cellof claim 58, wherein the immune cell has disrupted NKG2A, CD47, TGFβR2,and CISH.
 76. The cell of claim 58, wherein the immune cell hasdisrupted NKG2A, CISH, TGFβR2 and ADORA2.
 77. The cell of claim 58,wherein the immune cell has disrupted NKG2A, TGFβR2 and CISH.
 78. Thecell of claim 58, wherein the immune cell has disrupted TIGIT, CD96,CISH, and ADORA2.
 79. The cell of claim 58, wherein the immune cell hasdisrupted ADAM17, TGFβR2 NKG2A and SHP1.
 80. The cell of claim 58,wherein the immune cell has enhanced antitumor cytotoxicity, in vivoproliferation, in vivo persistence, and/or improved function.
 81. Thecell of claim 58, wherein the immune cell has increased secretion ofIFN-γ, CD107, and/or TNFα.
 82. The cell of claim 58, wherein the immunecell has increased production of perforin and/or granzyme B.
 83. Thecell of claim 58, wherein the cell is engineered to express a CAR and/orTCR.
 84. The cell of claim 83, wherein the CAR is inserted at anendogenous inhibitory gene locus of said cell.
 85. The cell of claim 84,wherein the inhibitory gene locus is selected from the group consistingof NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96,ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6,4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7.86. The cell of claim 84, wherein the CAR is under the control of theendogenous promoter of said inhibitory gene.
 87. The cell of claim 84,wherein the CAR was inserted at the inhibitory gene locus byCRISPR-mediated gene editing.
 88. The cell of any one of claims 83-87,wherein the CAR comprises an antigen-binding domain selected from thegroup consisting of F(ab′)2, Fab′, Fab, Fv, and scFv.
 89. The cell ofany one of claims 83-88, wherein the CAR targets one or more tumorassociated antigens selected from the group consisting of CD19, CD319(CS1), ROR1, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125,MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutatedp53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5,CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha,kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33,CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, and CD99.
 90. The cell ofany one of claims 83-90, wherein the CAR comprises at least onesignaling domain selected from the group consisting of CD3, CD28,OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132,DAP12, CD70, and CD40.
 91. The cell of any one of claims 58-90, whereinthe cell is engineered to express a heterologous cytokine selected fromthe group consisting of IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, and acombination thereof.
 92. The cell of claim 83, wherein the cell furthercomprises a suicide gene.
 93. The cell of claim 92, wherein the suicidegene is a membrane bound tumor necrosis factor (TNF)-alpha mutant gene.94. An expression vector encoding a CAR, inhibitory gene sequence, andgRNA.
 95. The vector of claim 94, wherein the inhibitory gene sequenceis from an inhibitory gene selected from the group consisting of NKG2A,SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1,PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40,IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7.
 96. The vector ofclaim 94, wherein the gRNA is specific to said inhibitory gene.
 97. Thevector of claim 94, wherein the vector is a retroviral vector.
 98. Thevector of claim 94, wherein the retroviral vector is an AAV vector. 99.The vector of claim 94, wherein the CAR is flanked by homology arms forthe inhibitory gene.
 100. A host cell engineered to express the vectorof any of claims 94-99.
 101. The cell of claim 100, wherein the cell isa T cell, NK cell, B cell, or stem cell.
 102. The cell of claim 100,wherein the cell is a cell of any one of claims 58-83.
 103. Apharmaceutical composition comprising a population of immune cells ofany one of claims 58-93.
 104. A composition comprising a population ofcells of any one of claims 58-93 for the treatment of an immune-relateddisorder, infectious disease, or cancer.
 105. A method of treating adisease or disorder in a subject comprising administering an effectiveamount of immune cells of any one of claims 58-93 to the subject. 106.The method of claim 105, wherein the disease or disorder is aninfectious disease, cancer, or immune-related disorder.
 107. The methodof claim 106, wherein the immune-related disorder is an autoimmunedisorder, graft versus host disease, allograft rejection, orinflammatory condition.
 108. The method of claim 106, wherein theimmune-related disorder is an inflammatory condition and the immunecells have essentially no expression of glucocorticoid receptor. 109.The method of any one of claims 105-108, wherein the immune cells areautologous to the subject.
 110. The method of any one of claims 105-108,wherein the immune cells are allogeneic to the subject.
 111. The methodof claim 106, wherein the immune-related disorder is a cancer.
 112. Themethod of claim 111, wherein the cancer is a solid cancer or ahematologic malignancy.
 113. The method of any one of claims 105-112,further comprising administering to the subject at least a secondtherapeutic agent.
 114. The method of claim 113, wherein the at least asecond therapeutic agent comprises chemotherapy, immunotherapy, surgery,radiotherapy, or biotherapy.
 115. The method of claim 113 or 114,wherein the immune cells and/or the at least a second therapeutic agentare administered intravenously, intraperitoneally, intratracheally,intratumorally, intramuscularly, endoscopically, intralesionally,percutaneously, subcutaneously, regionally, or by direct injection orperfusion.
 116. A method for engineering an immune cell to express aCAR, said method comprising using a CRISPR gRNA to insert the CAR at aninhibitory gene locus of said immune cell.
 117. The method of claim 116,wherein the CAR is encoded by an expression vector.
 118. The method ofclaim 117, wherein the expression vector is a retroviral vector. 119.The method of claim 118, wherein the retroviral vector is anadenovirus-associated vector.
 120. The method of claim 119, wherein theadenovirus-associated vector is AAV6.
 121. The method of claim 117,wherein the vector further comprises an inhibitory gene sequence. 122.The method of claim 121, wherein the inhibitory gene sequence is from aninhibitory gene selected from the group consisting of NKG2A, SIGLEC-7,LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1,PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R,ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7.
 123. The method of any oneof claims 116-122, wherein the CRISPR gRNA is to said inhibitory gene.124. The method of any one of claims 116-123, wherein the CAR is flankedby homology arms for said inhibitory gene.
 125. The method of any one ofclaims 116-124, wherein the CAR is inserted at an exon of saidinhibitory gene.
 126. The method of any one of claims 116-125, whereinthe CAR is under the control of the endogenous promoter of theinhibitory gene.
 127. The method of any one of claims 116-126, whereinthe CAR disrupts the expression of said inhibitory gene.
 128. The methodof any one of claims 116-127, wherein the CAR targets one or more tumorassociated antigens selected from the group consisting of CD19, CD319(CS1), ROR1, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125,MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutatedp53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5,CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha,kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33,CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, and CD99.
 129. The methodof any one of claims 116-128, wherein the CAR comprises at least onesignaling domain selected from the group consisting of CD3, CD28,OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132,DAP12, CD70, CD40, and a combination thereof.
 130. The method of any oneof claims 116-129, wherein the cell is engineered to express at leastone heterologous cytokine selected from the group consisting of IL-7,IL-2, IL-15, IL-12, IL-18, IL-21, and a combination thereof.
 131. Themethod of any one of claims 116-130, wherein the cell further comprisesa suicide gene.
 132. The method of claim 131, wherein the suicide geneis a membrane bound tumor necrosis factor (TNF)-alpha mutant gene. 133.An immune cell with a CAR inserted at an inhibitory gene of said immunecell.
 134. The immune cell of claim 133, wherein the cell is produced bythe method of any one of claims 116-132.
 135. A composition comprising apopulation of immune cells of claim 133 or
 134. 136. The composition ofclaim 135, wherein the immune cell is a T cell, B cell, or NK cell. 137.A composition comprising a population of cells of any one of claims133-136 for the treatment of an immune-related disorder, infectiousdisease, or cancer.
 138. A method of treating a disease or disorder in asubject comprising administering an effective amount of cells of any oneof claim 58-93, 100, or 133-134 to the subject.
 139. The method of claim138, wherein the disease or disorder is an infectious disease, cancer orimmune-related disorder.
 140. The method of claim 139, wherein theimmune-related disorder is a autoimmune disorder, graft versus hostdisease, allograft rejection, or inflammatory condition.
 141. The methodof claim 139, wherein the immune-related disorder is an inflammatorycondition and the immune cells have essentially no expression ofglucocorticoid receptor.
 142. The method of any one of claims 138-141,wherein the cells are autologous for the subject.
 143. The method of anyone of claims 138-141, wherein the cells are allogeneic.
 144. The methodof claim 138, wherein the immune-related disorder is a cancer.
 145. Themethod of claim 144, wherein the cancer is a solid cancer or ahematologic malignancy.
 146. The method of any one of claims 138-145,further comprising administering to the subject at least a secondtherapeutic agent.
 147. The method of claim 146, wherein the at least asecond therapeutic agent comprises chemotherapy, immunotherapy, surgery,radiotherapy, or biotherapy.
 148. The method of claim 146 or 147,wherein the immune cells and/or the at least a second therapeutic agentare administered intravenously, intraperitoneally, intratracheally,intratumorally, intramuscularly, endoscopically, intralesionally,percutaneously, subcutaneously, regionally, or by direct injection orperfusion.